copy 2009 Maulik P Suthar

Screening for microbes and strain improvement

Maulik P Suthar

copy 2009 Maulik P Suthar

Microbial products

bull Primary metabolites Made during the organismrsquos growth phase Essential to an organismrsquos metabolism and can be intermediate metabolites or end

bull Secondary metabolites Not essential to cell function or growth and are usually made late in the organismrsquos growth cycle Usually derived from primary metabolites or intermediates of primary metabolites Most likely give the organism an advantage over competitors

Primary Secondary

copy 2009 Maulik P Suthar

Methods for screening

bull Microbial bull Genotypicbull Phenotypic bull Chemical

copy 2009 Maulik P Suthar

Strain improvement

bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering

bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre

copy 2009 Maulik P Suthar

What are Antibiotics

bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi

from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic

chemicalsbull Most of the antibiotics identified over the past 60 years have been

natural products produced by one micro-organism within a particular environment to affect neighbouring microbes

bull Can cause microbe death or regulate the growth of the neighbouring microbes

bull These antibiotic agents are produced by both bacteria and fungi

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Microbial products

bull Primary metabolites Made during the organismrsquos growth phase Essential to an organismrsquos metabolism and can be intermediate metabolites or end

bull Secondary metabolites Not essential to cell function or growth and are usually made late in the organismrsquos growth cycle Usually derived from primary metabolites or intermediates of primary metabolites Most likely give the organism an advantage over competitors

Primary Secondary

copy 2009 Maulik P Suthar

Methods for screening

bull Microbial bull Genotypicbull Phenotypic bull Chemical

copy 2009 Maulik P Suthar

Strain improvement

bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering

bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre

copy 2009 Maulik P Suthar

What are Antibiotics

bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi

from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic

chemicalsbull Most of the antibiotics identified over the past 60 years have been

natural products produced by one micro-organism within a particular environment to affect neighbouring microbes

bull Can cause microbe death or regulate the growth of the neighbouring microbes

bull These antibiotic agents are produced by both bacteria and fungi

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Methods for screening

bull Microbial bull Genotypicbull Phenotypic bull Chemical

copy 2009 Maulik P Suthar

Strain improvement

bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering

bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre

copy 2009 Maulik P Suthar

What are Antibiotics

bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi

from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic

chemicalsbull Most of the antibiotics identified over the past 60 years have been

natural products produced by one micro-organism within a particular environment to affect neighbouring microbes

bull Can cause microbe death or regulate the growth of the neighbouring microbes

bull These antibiotic agents are produced by both bacteria and fungi

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Strain improvement

bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering

bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre

copy 2009 Maulik P Suthar

What are Antibiotics

bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi

from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic

chemicalsbull Most of the antibiotics identified over the past 60 years have been

natural products produced by one micro-organism within a particular environment to affect neighbouring microbes

bull Can cause microbe death or regulate the growth of the neighbouring microbes

bull These antibiotic agents are produced by both bacteria and fungi

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

What are Antibiotics

bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi

from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic

chemicalsbull Most of the antibiotics identified over the past 60 years have been

natural products produced by one micro-organism within a particular environment to affect neighbouring microbes

bull Can cause microbe death or regulate the growth of the neighbouring microbes

bull These antibiotic agents are produced by both bacteria and fungi

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antibiotic Production

bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts

bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population

bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Mutations and Genetic Instability

bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Induced Mutations

UV radiation

Chemical Mutations

1 4-Nitroquinoline-1-oxide

2 Hydroxylamine

3 Methyl Methanesulfonate

4 Ethyl Methanesulfonate

5 N-methyl-N-nitro-N-nytrosoguanidine

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today

1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Plate Based Techniques

bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic

diffused into the surrounding agar

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antibiotic Production

bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value

Why screening bull In order to identify the useful antibiotics a process of screening is

often employed bull Using this method isolates of a large number of micro-organisms

are cultured and then tested for production of diffusible products which inhibit the growth of test organisms

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Production with microbes

Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and

maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Steps in Industrial fermentation

Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)

(raw material)

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Industrial organism

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

AN INDUSTRIAL MICROORGANISM MUST

1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity

(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the

substratendash COMMODITY chemicals are inexpensive chemicals produced in

bulk including ethanol citric acid and many others - DEE antibiotics)

3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable

4 Be non-pathogenic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Industrial micro-organisms

bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can

be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one

or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism

found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other

industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)

bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antibiotic producing organisms

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Level of antimicrobial activity

bull Effectiveness expressed in two ways

1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen

- Dilution of series of antimicrobial compounds made

- Lowest conc That inhibits is MIC

2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen

- Subculturing into fresh medium can reveal MLC

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antimicrobial tests

bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug

bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on

agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to

compound

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

The Etest- strip test

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

How do the microbes produce theseproducts

bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express

this genes in microbes used in productionbull Able to produce high amount of compounds than in original

organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material

for fermentation

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Search for new antibiotics

Two approaches

1 Bioprospecting

2 Use of molecular microbiology computational chemistry

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

ndash Soil organisms

ndash streptomycetes actinomycetes fungi

ndash Marine actinomycetes

ndash Plants

ndash Corals and other marine organisms

ndash Products from insects and animals eg frog skin secretions

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Bioprospecting - Step 1

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Bioprospecting - step 2

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Molecular approaches

bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules

bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH

bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

DNA microarrays

bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)

bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions

bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in

the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action

(MOA) or identify targets with unknown MOA

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

The Design Bicycle

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Novel approaches

bull Virtual screeningndash Enzyme purification crystallization and 3-D structural

determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can

be identified using computer modellingbull Approach only works for targets amenable to 3-D structure

determination ndash many targets are too complexndash Various known or unknown structures docked to target site and

those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a

number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Novel approaches

2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S

aureusbull Using microarray methods - estimated that genome contained 265-359 essential

genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets

ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro

ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli

bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential

ndash Random mutagenesisndash Targeted mutagenesis

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Identification of targets

bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system

(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse

low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition

and enzyme spectrum and selectivity

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Genomics and target basedidentification of new antimicrobials

bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with

respect to optimizing potency spectrum and selectivity

bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 1 ndash primary screen

Elementary tests to determine activity in-vitro against small number of key organisms

bull Stage 2 - secondary screen

Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine

bull Stage 3 ndash toxicity testing

Compounds thoroughly tested in animals

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Search for new antibiotics

bull Stage 4 ndash human pharmacokinetics

Measurement of distribution of the compound in the human body

Potential dosage regimes and routes of administrationbull Stage 5 clinical trials

Effectiveness amp acceptability of the compound tested in patients

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the

compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Parameters which determine theusefulness of an antibiotic

bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration

by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via

various routesbull Side effects in volunteers ndash pain on injection nausea headache or

other symptoms not predicted by toxicity tests in animals

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antibiotic Targets

bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis

ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)

(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)

(3) DNA replicationndash quinolones(Cipro)

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria

(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked

peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis

-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome

-stop elongation of growing peptide chains(3) DNA replication

-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic

copy 2009 Maulik P Suthar

Questions

bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and

semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil

organismsbull Describe the importance of molecular advances in the search for

new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new

antibiotic