0 Med Chem, chapter 9.ppt - Cal Poly Pomonapsbeauchamp/pdf/499_Med_Chem_topic... · 2017-05-10 · Mechanism of action • Contain planar aromatic or heteroaromatic ring systems

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Types of DRUGS ACTING on DNA (in chapter 9)

Intercalating agents

Topoisomerase poisons

Chap. 9 - DRUGS TARGETING NUCLEIC ACIDS DNA & RNA

Alkylating agents

Metallating agents

Chain cutters

Chain terminators

© Oxford University Press, 2013

Chain terminators

Control of gene transcription (and epigenetics)

1

DRUGS ACTING ON DNA

Intercalating agents

Mechanism of action

• Contain planar aromatic or heteroaromatic ring systems• Planar systems slip between the layers of nucleic acid pairs and

disrupt the shape of the helix• A preference is often shown for either the minor or major groove• Intercalation prevents replication and transcription • Intercalation can inhibit topoisomerases too


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Intercalating agentsProflavine, also called diaminoacridine, is an acriflavine derivative, a disinfectant bacteriostatic against many gram-positive bacteria. It has been used in the form of the dihydrochloride and hemisulfate salts as a topical antiseptic, and was formerly used as a urinary antiseptic. Proflavine is toxic and carcinogenic in mammals and so it is used only as a surface disinfectant or for treating superficial wounds.

Proflavine is also known to have a mutagenic effect on DNA byProflavine is also known to have a mutagenic effect on DNA byintercalating between nucleic acid base pairs. It differs from most other mutagenic components by causing basepair-deletions or basepair-insertions and not substitutions.

NH2N NH2

Ar

NH3 Cl

HOSO3

NH3 Cl

NH3


Planar tricyclic systemThe amino substituents are protonated Used as a topical antibacterial agent in WW IITargets bacterial DNAToo toxic for systemic use

HOSO3

Ar

NH3

NH3

as a salt proflavine is more stable and avoids oxidation

HOSO3

sugar phosphateNH3N NH3

Proflavine

A

G C

T

CGCG

ATAT

Intercalating agents: Proflavine

T A

G C

H3N NH3

O O

proflavine

sugar phosphatebackbone

T

TAG

C

ATC

GCTA

ATCG

A

T AT A

TA

TAT A

G C

C

T ATA

CG

G

G


Ionic interactions

DNA DOUBLE HELIX

van der Waals interactions

4

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N

ethidium bromide

Br

Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. It has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle, a disease caused by trypanosomes. The high incidence of antibiotic resistance makes this treatment impractical in

H2N NH2

antibiotic resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Ethidium bromide may be a mutagen, although this depends on the organism exposed and the circumstances of exposure. Approximately 30,000 people in sub-Saharan Africa get African trypanosomiasis each year and Chagas disease, which causes 21,000 deaths per year mainly in Latin America.

fluorescent stain for gel electrophoresis

© Oxford University Press, 20135Protozoan trypanosomes spread by the Tsetse Fly, develops over years

N

N NH2

Br

NN

HNH

H2N

isometamidium chloride

Isometamidium is a phenanthridine aromatic amidine with a narrow therapeutic index which has been marketed for over 30 years as both a prophylactic and a therapeutic trypanocidal agent in the field. Isometamidium chloride is used curatively at lower dosage rates, and prophylactically at higher dosage rates.

Indications


IndicationsIntromidium is indicated for treatment and prevention of trypanosomiasis caused by Trypanosoma spp. in cattle, goats, sheep, camels, horses and dogs. A preventive dosage ensures protection for 2 to 4 months. When clinical cases occur, the whole group should be treated.

Contra indicationsIntromidium should not be administered subcutaneously.Avoid concurrent administration of other trypanocidal drugs, particularly diminazene aceturate. Avoid underdosing.

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Planar rings

Doxorubicin (also called adriamycin)1. Intercalates via the major groove of DNA double helix.2. Blocks the action of topoisomerase II by stabilising the DNA-enzyme complex 3. Acts as a topoisomerase poison

Doxorubicin is commonly used to treat some leukemias and

Intercalating agents - anticancer agents

OMe

O

O

OH

OH H O

OH

O

CH2OH

OH

MeH

gDoxorubicin is commonly used to treat some leukemias and Hodgkin's lymphoma, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, and others Many combination therapies are used, such as (adriamycin, cyclophosphamide),

Liposomal formThere is a pegylated (polyethylene glycol coated) liposome-encapsulated form of doxorubicin, sold as Doxil. Doxil is used primarily for the treatment of ovarian cancer or AIDS-related


NH3

H

H

H

Me

HOH

Doxorubicin (Adriamycin)

Extra binding to sugar phosphate backbone by NH3

Kaposi's sarcoma where the disease has progressed or recurred after platinum-based chemotherapy.

O NHP

O NCl

Cl Cyclophosphamide Common side effects include low white blood cell counts, loss of appetite, vomiting, hair loss, bleeding from the bladder, an increased future risk of cancer, infertility, allergic reactions, and pulmonary fibrosis. Cyclophosphamide is in the alkylating agent and nitrogen mustard family of medications. It works by interfering with the duplication of DNA and the creation of RNA.

The biosynthesis of Doxorubicin uses enzymes found in fat metabolism. This involves using acetyl building blocks (2C) as acetyl CoA. Such molecules are called “kitids” (often multiples of 2C). There are 1000s of compounds that have been discovered from the random synthesis of clusters of proteins working together. Chemists have tried to splice in various combinations of such genes in microorganisms to make novel drugs as potential lead compounds


compounds.

a few more steps...

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Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. A comparison of uricase and PEG-uricase. PEG-uricase includes 40 polymers of 10kDa PEG. PEGylation improves its solubility at physiological pH, increases serum half-life and reduces immunogenicity without compromising activity. Upper images show the whole tetramer, lower images show one of the lysines that is PEGylated.

O

Nu

O

O

OO

OO

etc.

Nu

OH

OH2N O

OH

NO

OHpH = 5

( H O)


O

OHO H

nO

OH

Fe+3

n

imine (oxime) linkage to the drug through a carbonyl group

(-H2O)

O

OH

O

OH

HO

OH

n

phosgene or phosgene equivalent

Cl Cl

O

O

OH

O

O Cl

O H

O

OH

O

O

O

OO

H

n

Dactinomycin1. Intercalates via minor groove of DNA double helix2. Prevents unwinding of DNA double helix3. Blocks transcription by blocking DNA-dependent RNA polymerase

Dactinomycin is a chemotherapy used to treat i l di Wil t

Intercalating agents - anticancer agents

many cancers, including Wilms tumor, rhabdomyosarcoma, Ewing's sarcoma, trophoblastic neoplasm, testicular cancer, and certain types of ovarian cancer. It is given by injection into a vein. Common side effects include bone marrow suppression, vomiting, mouth ulcers, hair loss, liver problems, infections, muscle pains, future cancers, allergic reactions, and tissue death at the site of injection. Use in pregnancy may harm the baby.

Dactinomycinplanar rings

can intercallate


Extra binding to sugar phosphate backbone by cyclic peptides

injection. Use in pregnancy may harm the baby.

Dactinomycin. also known as actinomycin D, is the most significant member of actinomycines, which are a class of polypeptide antitumor antibiotics isolated from soil bacteria of the genus Streptomyces. It is one of the older anticancer drugs, and has been used for many years.

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Many quinone methides show medicinal properties

Can be extended to naphthalene, anthracene, phenanthrene

Taxodone and its oxidized rearrangement product, taxodione, are diterpenoid quinone methides found in Taxodium distichum (bald cypress), Rosmarinus officinalis (rosemary), several Salvia species and other plants, that display anticancer, antibacterial, antioxidant, antifungal insecticide and antifeedant activities

O O

para quinone methide pattern

ortho quinone methide pattern

two common patterns

B antifungal, insecticide, and antifeedant activities.p p

OH

O

HO

oxidationlose proton

H

H

1e-O

O

HO

H

H

resonance

oxidationlose proton

1e- O

O

HO

H

H

H

BTaxodone


O

O

HO

H

RH2N

BHOH

O

HO

N

H

HR

nucleophiles undergo

conjugate addition

Intercalating agents and chain cutting - Bleomycins

N N

CONH2HN

NH2

NH2

O

H NO

OHO

MeHN

NH

O

N

SN

R

OH

H

H H Hpyrimidinering

primary amine

primary amide

Bithiazole

N N

CONH2HN

NH2

NH2

O

H NO

OHO

MeHN

NH

O

N

SN

R

OH

H

H H Hpyrimidinering

primary amine

primary amide

Fe+2 or Fe+3 can chelate here

A variety of possibilities.

H2N

Me HNNH

O

MeO

HO Me

NH

S

N

O

NH

N

O

HO

OH

OH

O

OH

H

H

H H

Bleomycin A2 R = NHCH2CH2CH2SMe2Bleomycin B2 R = NHCH2CH2CH2CH2NHC(NH2)=NH

imidazolering

primary amine

Bithiazoleintercalatingregion

H2N

Me HNNH

O

MeO

HO Me

NH

S

N

O

NH

N

O

HO

OH

OH

O

OH

H

H

H H

Bleomycin A2 R = NHCH2CH2CH2SMe2Bleomycin B2 R = NHCH2CH2CH2CH2NHC(NH2)=NH

imidazolering

primary amine

Notes on bleomycins


OHO

NH2

O

OH OHO

NH2

O

OH

12

y• Used as anticancer agents• Intercalated by means of bithiazole ring system• Ferrous ion then chelated by nitrogens of the primary

amines, amide and pyrimidine ring• Reaction with oxygen results in a ferric ion and reactive

oxygen species• Results in radical formation and chain cutting• Bleomycin prevents DNA ligase from repairing damage

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Bleomycin is a medication used to treat cancer. This includes Hodgkin's lymphoma, non-Hodgkin's lymphoma, testicular cancer, ovarian cancer, and cervical cancer among others. Typically used with other cancer medications, it can be given intravenously, by injection into a muscle or under the skin. It may also be administered inside the chest to help prevent the recurrence of a fluid around the lung due to cancer; however talc is better for this.

Common side effects include fever, weight loss, vomiting, rash, a severe type of anaphylaxis, possible inflammation of the lungs that can result in lung scarring Chest X rays every couple of


in lung scarring. Chest X-rays every couple of weeks are recommended to check for this. Bleomycin may cause harm to the baby if used during pregnancy. It is believed to primarily work by preventing synthesis of DNA.

OO

O

Base

H

DNA

O

OO

O

Base

DNA

OO

O

Base

O

DNA

H B

BH

Free radical damage to DNA can remove bases and

OH

Fe+3

HN NHDNA

Fe+4 DNA DNA

BaseH

OO

DNA

O

Iron is believed to be the metal responsible for the creation of free radicals (often hydroxyl) because it exists at the highest concentration of any transition metal in most living organisms.

Free radicals can attack the deoxyribose DNA backbone and

HN NH

Bleomycin

Fe+4

HN NH

Bleomycin

HN NH

Bleomycin


O

DNA BH

ybases, potentially causing a number of lesions that can be cytotoxic or mutagenic. Cells have developed complex and efficient repair mechanisms to fix the lesions. In the case of free radical attack on DNA, base-excision repair is the repair mechanism used. Free radical reactions with the deoxyribose sugar backbone (C1, C2, C3, C4 or C5) are initiated by hydrogen abstraction from a deoxyribose carbon, and the predominant consequence is eventual strand breakage and base release.

DNA strands can unravel.

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Topoisomerase medications are split into two main classes: topoisomerase poisons, which target the topoisomerase-DNA complex, and topoisomerase inhibitors, which disrupt catalytic turnover.

Topo II poisons

Examples of topoisomerase poisons include the following:eukaryotic type II topoisomerase inhibitors (topo II): amsacrine, etoposide (next slide), etoposide phosphate, teniposide and doxorubicin. These drugs are anti-cancer therapies.b t i l t II t i i hibit ( d t IV) fl i l Thbacterial type II topoisomerase inhibitors (gyrase and topo IV): fluoroquinolones. These are antibacterials and include such fluoroquinolones as ciprofloxacin.

Some of these poisons encourage the forward cleavage reaction (fluoroquinolones), while other poisons prevent the re-ligation of DNA (etoposide and teniposide).

Ciprofloxacin (later slides) targets prokaryotes topo II a thousand times better than it targets eukaryotic topo IIs.

Topo II inhibitors

O

H


Topo II inhibitors

These inhibitors target the N-terminal ATPase domain of topo II and prevent topo II from turning over. Very few of these inhibitors.

An example of topoisomerase inhibitors includes :ICRF-193. The structure of this compound bound to the ATPase domain is known. The drug binds in a non-competitive manner and locks down the dimerization of the ATPase domain.

N

NN

N

O

O

O

H

H ICRF 193

Topoisomerase poisons - non-intercalating

O

O

O

O

OO

OHO

Me

H

HO

4

Stabilizes the complex between DNA and topoisomerase enzymes Used as anticancer agents Also causes chain cutting

MeO

OH

OMe

O

4'

Etoposide

Etoposide is used as a form of chemotherapy for cancers such as Kaposi’s sarcoma, Ewing's sarcoma, lung cancer, testicular cancer, lymphoma, nonlymphocytic leukemia, and glioblastoma multiforme. It is often given in combination with other drugs (such as bleomycin). It is also sometimes used in a conditioning regimen prior to a bone marrow or blood stem cell transplant.


It is given intravenously (IV) or orally in capsule form. If the drug is given IV, it must be done slowly over a 30- to 60-minute period because it can lower blood pressure as it is being administered. Blood pressure is checked often during infusing, with the speed of administration adjusted accordingly.

Etoposide forms a ternary complex (3 way) with DNA and the topoisomerase II enzyme (which aids in DNA unwinding), prevents re-ligation of the DNA strands, and by doing so causes DNA strands to break. Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. This causes errors in DNA synthesis and promotes apoptosis of the cancer cell.

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O

O

O

O

OO

OHO

H

HO

4

S

Stabilizes the complex between DNA and topoisomerase enzymesUsed as anticancer agentsAlso causes chain cutting, similar to etoposide.

O

MeO

OH

OMe

O

4'

Teniposide

Teniposide is a chemotherapeutic medication used in the treatment of childhood acute lymphocytic leukemia, Hodgkin's lymphoma, certain brain tumours, and other types of cancer. It slows the growth of cancer cells in the body (by a ternary complex).


When Teniposide is used with other chemotherapeutic agents, it results in severe bone marrow suppression. Other common side effects include gastrointestinal toxicity, hypersensitivity reactions, and reversible alopecia (hair loss).

Teniposide is a semisynthetic derivative of podophyllotoxin from the rhizome of the wild mandrake(Podophyllum peltatum). More specifically, it is a glycoside of podophyllotoxin with a D-glucose derivative. It is chemically similar to the anti-cancer drug etoposide, being distinguished only by a thienyl where etoposide has a methyl. Both these compounds have been developed with the aim of creating less toxic derivatives of podophyllotoxin.

DRUGS ACTING ON DNA - Topoisomerase poisons - non-intercalating

Examples - Camptothecin Notes• Stabilizes a complex between DNA and

topoisomerase I• Single-strand breaks accumulate in the chain• Irreversible double-strand breaks occur during

transcription

N

N

O

OA B C

D

E Lactone ringtranscription

• Semi-synthetic analogues used as anticancer agents

OHOMe

Camptothecin is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I). It was isolated from the bark and stem of Camptotheca acuminata (Happy tree), a tree native to China and used as a cancer treatment in Traditional Chinese Medicine. It showed remarkable anticancer activity in preliminary li i l i l b l l l bili d (hi h) d d i B f h


clinical trials but also low solubility and (high) adverse drug reaction. Because of these disadvantages synthetic and medicinal chemists have developed numerous syntheses of Camptothecin and various derivatives to increase the benefits of the chemical, with good results. Two analogues have been approved and are used in cancer chemotherapy today, topotecan and irinotecan.

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Camptothecin, DNA and enzyme complex Camptothecin binds to the topo I enzyme and DNA complex by hydrogen bonds resulting in a ternary complex, and thereby stabilizing it. This prevents DNA re-ligation and causes DNA damage which results in apoptosis. The most important part of the structure is the E-ring which interacts from three different positions with the enzyme. The hydroxyl

DNA

ProteinA B C group in position 20 forms hydrogen bond to

the side chain on aspartic acid number 533 in the enzyme. It is critical that the configuration of the chiral carbon is (S) because (R) is inactive. The lactone is bonded with two hydrogen bonds to the amino groups on arginine 364. Toxicity of CPT is primarily a result of conversion of single-strand breaks into double-strand breaks during the S-phase when the replication fork collides with the

Drug

A B C

D

E

RR


when the replication fork collides with the cleavage complexes formed by DNA and CPT. The lactone ring in CPT is highly susceptible to hydrolysis. The open ring form is inactive and it must therefore be closed to inhibit topo I. The closed form is favored in acidic condition, as it is in many cancer cells microenvironment. CPT is transported into the cell by passive diffusion.

N

N

O

O

OOH

R1R2

R3

R4

There are many variations made with different groups at R1, R2, R3 and R4

N

N

O

O

O

N

N

O

O

O

HO

N

Topotecan (trade name Hycamtin) is a chemotherapeutic agent that is a topoisomerase I inhibitor. It is a synthetic, water-soluble analog of the natural chemical compound camptothecin. It is used in the form of its hydrochloride salt to treat ovarian cancer, lung cancer and other cancer types. Topotecan was the first topoisomerase I inhibitor for oral use. Why is it more water soluble? Loperamide often taken with topotecan due to severe diarhea side effect.

TopotecanCamptothecin

SN 38 i i l i d I i h i b li f i i (

N

N

O

O

O

irinotecan(next slide)

N

N

O

ON

N

O

O

O

HO

SN-38

SN-38 is an antineoplastic drug. It is the active metabolite of irinotecan (an analog of camptothecin - a topoisomerase I inhibitor) but has 1000 times more activity than irinotecan itself. SN-38 and its glucuronide are lost into the bile and feces. It can cause the symptoms of diarrhoea and myelosuppression experienced by ~25% of the patients administered irinotecan.


O

NOH

O N

Cl

Loperamide, sold under the brand name Imodium among others, is a medication used to decrease the frequency of diarrhea. It is often used for this purpose in gastroenteritis, inflammatory bowel disease, and short bowel syndrome. It is not recommended for those with blood in the stool. The medication is taken by mouth.

Loperamide

O

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Model of human liver cell showing blood, bile and intestinal compartments, indicating tissue involvement of genes in the irinotecan pathway – eventually leads to inhibition of DNA replication and transcription.

Irinotecan-associated diarrhea sometimes leads to such severe dehydration that it requires hospitalization. This side-effect is t t d ith tidi h l htreated with an antidiarrheal such as loperamide.

The immune system is adversely impacted by irinotecan, reflected by dramatically lowered neutrophils (white blood cells). The patient may experience a period of neutropenia while the bone marrow increases white cell production to compensate.

Neutrophils make up the majority of


21

p p j ycirculating white blood cells and serve as the primary defense against infections by destroying bacteria, bacterial fragments and immunoglobulin-bound viruses in the blood. Patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening (neutropenic sepsis). Neutropenia can be acute (temporary) or chronic (long lasting).

The partition-coefficient (P) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. This ratio is a measure of the difference in solubility of the compound in these two phases. The partition-coefficient generally refers to the concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration ratio of all species of the compound (ionized plus un-ionized) and depends on pH. Usually one phase is water and the other is 1-octanol. The partition coefficient measures how hydrophilic or hydrophobic a chemical substance is. Hydrophobic drugs with high octanol/water partition coefficients are mainly distributed to hydrophobic areas such as lipid bilayers of cells, while low octanol/water partition coefficients) are found primarily in aqueous regions such as blood serum.

log Poct/H2O = log [neutral compound]1-octanol[neutral compound]water

log Doct/H2O = log [all forms compound]1-octanol[all forms compound]water

solubility and partition coefficients of straight chain functiona groups in 100 g water

OHOH

OH OH

C5H11OH C6H13OH C7H15OH C8H17OH C9H19OHCH3

100% miscible

0.11 mol7.9g

0.030 mol2.6g

0.0058 mol0.6g

0.0008 mol0.09g

log Poct/H2O =

100% miscible

100% miscible

solubility =

-0.74 -0.38 +0.25 +0.84 +1.51 +2.03 +2.62

C5H11OH C6H13OH C7H15OH

0.0058 mol0.6g

0.0008 mol0.09g

+2.97 +4.02

C8H17OH C9H19OH

H3CC

O

C

O

100% miscible

4.9g100% miscible

100% misciblesolubility =

0 33 0 79 +1 39 +1 92 +2 6 +3 05

OH OH C2H5

C

O

OH C3H7

C

O

OH C4H9

C

O

OH C5H11

C

O

OH C6H13

C

O

OH C7H15

C

O

OHH

100% miscible

1.1g 0.2g(guess)

0.07g


log Poct/H2O = -0.54 -0.32 +0.33 +0.79 +1.39 +1.92 +2.6(guess)

+3.05

O

C

O

100% miscible

log Poct/H2O =

100% miscible

100% misciblesolubility =

+0.35 +0.45 +0.59 -0.24 +0.88 +0.29

H H

O

H

O O

H

O

H

100% miscible

27g soluble (?)

+1.3(guess)

+0.84

O

H

O

6g100% miscible

web site with lots of partition coef. values: https://www.nist.gov/sites/default/files/documents/srd/jpcrd367.pdf

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There is good correlation of calculated partition coefficients with experimental partition coefficients

log Poct/H2O = log [compound]1-octanol[compound]water

1.0

1.5

log D


What might a distribution coefficient curve look like for the following compound?

0.5

0.0

-0.5

-1.0

-1.5

pH2 4 6 8 10 12 14

OH

O

H2N


Examples - Quinolones and fluoroquinilones

N N

O

CH2CH3

CO2H

Me N

CO2H

N

O

HN

F

• Synthetic agents used as antibacterial agents• Stabilise complex between bacterial DNA and topoisomerases• Binding site for agents revealed once DNA strands are ‘nicked’

3

Nalidixic acid Ciprofloxacin

The quinolones are a family of synthetic broad-spectrum antibiotic drugs. They can be isolated from natural sources (such as plants, animals and bacteria) and act as natural antimicrobials.

Quinolones exert their antibacterial effect by preventing bacterial DNA from unwinding and


Quinolones exert their antibacterial effect by preventing bacterial DNA from unwinding and duplicating. The majority of quinolones in clinical use are fluoroquinolones, which have a fluorine atom attached to the central ring system, typically at the 6-position or C-7 position.

Fluoroquinolones are especially important for hospital-acquired infections in which resistance to older antibacterial classes is suspected. Treatment guidelines recommend minimizing the use of fluoroquinolones and other broad-spectrum antibiotics in less severe infections and in those in which risk factors for multidrug resistance are not present. Side effects are severe so quinolones are contraindicated unless no other safe alternative antibiotic exists.

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Tertiary Structure

1. Double helix coils into a 3D shape – supercoiling 2. Double helix has to unravel during replication 3. Unravelling leads to strain 4. Relieved by enzyme-catalyzed cutting and repair of DNA chain 5. Quinolone and fluoroquinolone antibacterial agents inhibit this enzyme

NH

Nalidixic acid (1962) was the first of the quinolone family of antibiotics.

The quinolones are a family of synthetic broad-spectrum antibiotic drugs. Quinolones, and derivatives, have also been isolated from natural sources (such as plants, animals and bacteria) and can act as natural antimicrobials and/or signalling molecules. Quinolones prevent bacterial DNA from unwinding and duplicating.

Ci fl i (1980) i d t t t b

NN CH3

OO

HO

Nalidixic acid (1962)

1

2

3

4 5

6

7

8

4a

8a

c


N N

OO

HOF

Ciprofloxacin (1980)

Ciprofloxacin (1980) is used to treat a number of bacterial infections including bone and joint infections, intra abdominal infections, certain type of infectious diarrhea, respiratory tract infections, skin infections, typhoid fever, and urinary tract infections, among others. A single F at positoin 6 greatly increased both activity and cellular uptake. The piperizine ring (ionized) at position 7 improved pharmacokinetics. Replacement of the N8 nitrogen reduced adverse reactions and increased activity agains S. aureus.

Which "N" is most basic? a, b, cd all are similar bases

a b


Examples - Quinolones and fluoroquinilones

TopoisomeraseTopoisomeraseenzyme

R6

R5 O

O

O

Region binding to DNA

R6

R5 O

O

ORegion binding t

Region R6

R5 O

O

O

R6

R5 O

O

ORegion binding t

Region R6

R5 O

O

O

Structure of bacterial DNA gyrase complexed with DNA and two ciprofloxacin molecules (green)


Fluoroquinolones

•Drug molecules are stacked in the bound complex• Bound to DNA and enzyme by hydrogen and ionic bonds

X8 NR7

R1

Stacking domain

X8 NR7

R1

to enzymebinding to enzyme

X8 NR7

R1

X8 NR7

R1

to enzymebinding to enzyme

X8 NR7

R1

26

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Topoisomerases I form a covalent intermediate in which the active site tyrosine becomes attached to the 3' phosphate end of the cleaved strand rather than the 5' phosphate end.

The TOP1 protein of humans has been subdivided into four regions. The N-terminal 214 amino acids are dispensable for relaxation of supercoiling activity in vitro. The N-terminal domain is followed by a highly conserved, 421 amino acid core domain containing all of the catalytic residues except the active site tyrosine. This is followed by a poorly conserved p y y p ylinker domain of 77 amino acids. Finally there is a 53 amino acid C-terminal domain. The active site Tyr723 is found within the C-terminal domain.

After the TOP1 covalently attaches to the 3' end of the broken strand, supercoiling of the DNA is relaxed by controlled rotation of DNA about the intact strand. Then the 5' hydroxyl end of the broken DNA strand can reverse the phosphotyrosyl bond, enabling the release of TOP1 and religation of the DNA. The nicking and closing reactions are fast, and about 100 cycles can occur per second.

Topoisomerase I

Topoisomerase II, shows DNA too


y p

One of the first inhibitors shown to target TOP1 is irinotecan. Irinotecan is an analogue of the cytotoxic natural alkaloid camptothecin, obtained from the Chinese tree Camptotheca acuminata. Irinotecan is especially effective through its metabolic product SN-38. Irinotecan and SN-38 act by trapping a subset of TOP1-DNA cleavage complexes, those with a guanine +1 in the DNA sequence. One irinotecan or SN-38 molecule stacks against the base pairs flanking the topoisomerase-induced cleavage site and poisons (inactivates) the TOP1 enzyme.

DRUGS ACTING ON DNA Alkylating agents

Nucleophilic atoms on nucleic acid bases

NN

NH2

NN

O

N

NH2

31

7H

nucleophilicatoms nucleophilic

atoms

5

nucleophilicatoms

N

N

N N

N

NH2N

R R

NO

R3

1

CytosineAdenine Guanine

nucleophilicatoms

1

electrophiles electrophiles electrophiles

NH2 O NH2E


N

NN

N

R

N

NN

NH2N

R

HN

NO

R

EE

https://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent

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• Contain highly electrophilic groups• Form covalent bonds to nucleophilic groups in DNA

(e.g. 7-N of guanine)• Prevent replication and transcription• Useful anticancer agents• Toxic side effects (e.g. alkylation of proteins)

Alkylating agents

( g y p )• Can cause interstrand and intrastrand crosslinking if two electrophilic groups present• Alkylation of nucleic acid bases can result in miscoding

Crosslinking is possible for doubly electrophilic compounds

NuNu Nu

X X

NuNu

X X

Nu

Nu


Interstrand crosslinkingIntrastrand crosslinking

NuNu

Cytosine Guanine

NH2 NO

Normal base pairing

Alkylating agents

Thymine Alkylated guanine

OMe NHO

DRUG

Miscoding resulting from alkylated nucleic acid bases

N

N

NH2

OR

HN

N

N

N

H2N

RN

NH

O

O

Me

R

N

N

N

NHO

R

H2N

Guanine prefers keto tautomer

Abnormal base pairing.Alkylated guanine prefers enol

tautomer


Normally, has 2 H-bonds with adenine Changes charge

and changes shape.

How many possible tautomers are there for DNA bases?

N

N

O

O

H

N

N

N

N

NH2

N

N

N

N

N

H

H

H

N

N

O

NHH

GuanineHHH

AdenineThymineCytosine

1

2

34

567

8

9 9

8

7 5

43

2

1

12

3

45

6

12

3

4

5

6

H

O

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Alkylating agents

Chlormethine (nitrogen mustard)

Me N

Cl

N

N

CH3

NCl

Bendamustine is a nitrogen mustard used in the treatment of chronic lymphocytic leukemia and lymphomas. It belongs to the family of drugs called alkylating agents. It is also being studied for the treatment of sarcoma. It is also being investigated for the non-cancer diseases.

• Secretly used medicinally in 1942 (WW II), reduced white blood count, many side effects, including increased cancer risk later

• Causes intrastrand and interstrand cross-linking• Prevents replication• Mono-alkylation of guanine also possible• Analogues with better properties have been prepared

Cl

NN

Cl


• Analogues with better properties have been prepared

31

Chlormethine is also used to synthesize pethidine. How might this be done? In 1975 pethidine was opiod of choice of 60% of doctors for acute pain and 22% for chronic severe pain

N

O

ON

Cl

Clchlormethine

?

pethidine

Cl

G = Guanine

DNA

HNO NH2

DNA

H

Mechanism of action

Alkylating agents

Chlormethine

How can DNA heal itself? Are there other possible leaving groups? How do they compare to chloride? Will they survive a journey through the body?

Mechlorethamine

H3C N

Cl

Cl Cl

NH3C

Aziridine ion

G

N

NN

N

N NH

N

O

NH2

G

N

N

N

NN

N

NH

N

O

NH2

O NH2

H3C N

Cl

DNA DNA


NN

N

N NH

N

O

NH2

N

HNO NH2

NH3C

CH3

N

N

N

N

N

N

HN

NH

N

O NH2

O

NH2

Crosslinked DNA

32

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Alkylating agents

Nitrosoureas

MechanismDecompose in body to form an alkylating agent and a carbamoylating agent

O

N NH

NO

Cl Cl

Carmustine

O

N NH

NO

Cl

Lomustine

Cl

N2 + HO

Alkylatingagent

ClN N

N

O

R

H

O C N R

ClN

+

Isocyanate(carbamoylating agent)

O C N RIsocyanate

Protein-Lys-NH2Protein-Lys-NH

O

HN RCarbamoylation


agentO

N

NOH

H O

33

ClDNA

X Y

Cl

X YCross-linking

DNA DNA

AlkylationAlkylatingagent

Notes• Alkylating agent causes interstrand crosslinking

• Crosslinking between G-G or G-C• Carbamoylating agent reacts with

lysine residues on proteins• May inactivate DNA repair enzymes

Alkylating agents

OO

S

O

CH3

S

O

O

H3C

MsOOMs=

Busulfan different length tethers are possible

Synthetic agent used as anticancer agent (dimesylate). Causes interstrand crosslinking

O

HN

N

O

N

N

H N

MsOOMs

N

N

OMs

N

N

N

DNA

SO

O

O

CH3

Very stable anionleaving group, could it be made even better?

Busulfan

Mechanism


NH2N

DNAGuanine N

DNAN

DNA

N

DNA

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Alkylating agents

• Prodrug activated by demethylation in liver• Decomposes to form a methyldiazonium ion• Alkylates guanine groups (methylates here)

HN N

CONH2NN

NH3C

CH

Dacarbazine

Mechanism

HN N

CONH2NN

NH3C

CH3

Cyt P-450

liver

HN N

CONH2NN

N

CH3OHH

HN N

CONH2NHN

N

CH3

-CH2O

H

HN N

CONH2H2N

N N CH3

AIC

Methyldiazonium ion

CH3


Methyldiazonium ion

N2 + CH3

35

other carbocations are possible

oxidation with cytochrom P-450

diazomethane (very toxic and explosive gas), usually made as basic ether solution using Diazald.

S

O O

N

N O

CH3

H O

N

N OH

CH2

HO

H

HH O

N

H2C

N

H

N

H2C

N

diazomethane

base

acid

Diazald

DRUGS ACTING ON DNA - Alkylating agents

Example - Mitomycin C

O

H2N

O

O

NH2

O CH

OH

H2N

NH

N

N

N

HN

DNA

O Guanine

• Prodrug activated in the body to form an alkylating agent• One of the most toxic anticancer drugs in clinical use

O

N NHH3C

2 O CH3

Where are the electrophilic sites?

How?

OH

NH3C

H2NHN

NH2 N

NN

HN

DNA

O

Guanine


• One of the most toxic anticancer drugs in clinical use

Mitomycin C is a mitomycin that is used as a chemotherapeutic agent by virtue of its antitumour activity. It is given intravenously to treat upper gastro-intestinal cancers (e.g. esophageal carcinoma), anal cancers, and breast cancers, as well as by bladder instillation for superficial bladder tumours. It causes delayed bone marrow toxicity and therefore it is usually administered at 6-weekly intervals. Prolonged use may result in permanent bone-marrow damage. It may also cause lung fibrosis and renal damage. 36

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Coenzyme Q10, also known as ubiquinone, is a coenzyme that is ubiquitous in the bodies of most animals. It is a 1,4-benzoquinone, where Q refers to the quinone chemical group and 10 refers to the number of isoprenyl chemical subunits in its tail. This fat-soluble substance, which resembles a vitamin, is present in most eukaryotic cells, primarily in the mitochondria. It is a component of the electron transport chain and participates in aerobic cellular respiration, which generates energy in the form of ATP. Ninety-five percent of the human body's energy is generated this way. There are three redox states of CoQ10: fully oxidized (ubiquinone), semiquinone, and fully reduced (ubiquinol). The capacity of this molecule to act as a two-electron carrier (moving between the quinone and quinol form) and a one-electron carrier (moving between the semiquinone and one of these other forms) is central to its role in the electron transport chain due to the iron–sulfur clusters that can only accept oneforms) is central to its role in the electron transport chain due to the iron–sulfur clusters that can only accept one electron at a time, and as a free radical-scavenging antioxidant.

O

O

O CH3

R

H3C

H3C

+one electron+one proton

B H

e-

O

O

O CH3

R

H3C

H3C

H

+one electron+one proton

e-

O

O

O

O CH3

R

H3C

H3C

H

both directions both directions


O

ubiquinone (CoQ10)oxidized

O

B H

Hubiquinol (CoQ10)

reducedsemiquinone (CoQ10)

a mycolic acid

O

O

N

H2N

Me

CH2OCONH2

NH

OMe

O

OH

N

H2N

Me

CH2OCONH2

NH

H

-MeOH

OH

OH

N

H2N

Me

CH2OCONH2

NH

OMe

ReductionNADH

O H

H

HN N

O Guanine

H2N-DNA

O

OH

N

H2N

Me

CH2OCONH2

NH2

Ringopening

-H +

Alkylating agent

OH

OH

N

H2N

Me

CH2

NH2

NH-DNA

OC NH2

H2N-DNA

H


OH

OH

N

H2N

Me

CH2

NH2

NH

NH

HN

N N

N

O

HN

N N

GuanineOH

OH

N

H2N

Me

CH2

NH2

NH-DNA

NH-DNA

-CO2

-NH3

Crosslinked DNA 38

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In the bacterium Legionella pneumophila, mitomycin C induces competence for transformation. Natural transformation is a process of DNA transfer between cells, and is regarded as a form of bacterial sexual interaction. In microbiology, genetics, cell biology, and molecular biology, competence is the ability of a cell to take up extracellular ("naked") DNA from its environment in the process called transformation. Competence may be differentiated between natural competence, a genetically specified ability of bacteria which is thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence, which arises when cells in laboratory cultures are treated to make them transiently permeable to DNA. In the natural world DNA usually becomes available by death and lysis of other cells, but in the laboratory it is provided by the researcher, often as a genetically engineered fragment or plasmid. During

Biosynthesis of mitomycins – working on a handout (how about you?)

, y p y , g y g g p guptake, DNA is transported across the cell membrane(s), and the cell wall if one is present. Once the DNA is inside the cell it may be degraded to nucleotides, which are reused for DNA replication and other metabolic functions. Alternatively it may be recombined into the cell’s genome by its DNA repair enzymes. If this recombination changes the cell’s genotype the cell is said to have been transformed.

OP

O

OH

O

H

OH

OH

OPO3-2

O

O

O

NH2O

CO2H

H

How?

CO2HHO

HO

OH

NH2

How?

CO2H

NH2

OH

HO

-2O3PO O

How?


aromatic intermediatePEP phosphoenolpyruvate

4-phosphoerythrose

O

H

NH2

OH

OH

OH

OH

glucosamine

NH2HO2C

OH

aromatic intermediate

How?

OH

NH3C

OH

OH

mitomycin C

OHamino DHQ

O

O

N NHH3C

H2N

O

O

NH2

O CH3

NH2

OH

mitosane core

How?

OH

amino DAHP

• Neutral inactive molecule acting as a prodrug• Platinum covalently linked to chloro substituents• Ammonia molecules act as ligands (RNH2)• Activated in cells with low chloride ion concentration• Chloro substituents replaced with neutral water ligands• Produces positively charged species• Binds to DNA in regions rich in guanine units • Intrastrand links rather than interstrand

L li d i di f DNA d bl h li

Metallating agents

DRUGS ACTING ON DNA

Pt

NH3Cl

NH3Cl

Cisplatin

• Localised unwinding of DNA double helix• Inhibits transcription

O

O

O

Pt

O

NH3

NH3

Carboplatin

Cisplatin, cisplatinum, platamin, neoplatin, cismaplat or cis-diamminedichloridoplatinum(II) (CDDP) is a chemotherapy drug. It was the first member of a class of platinum-containing anti-cancer drugs, which now also includes carboplatin and oxaliplatin. These platinum complexes react in the body, binding to DNA and causing the DNA strands to crosslink, which ultimately triggers cells to die in a programmed way.

Carboplatin, is a chemotherapy drug used against some forms of cancer (mainly ovarian carcinoma, lung, head and


PtNH3Cl

NH3Cl

H2OPt

NH3H2O

NH3ClPt

NH3H2O

NH3H2O

+ 2+

+DNA

PtNH3DNA

NH3DNA

Cisplatin40

neck cancers as well as endometrial, esophageal, bladder, breast and cervical; central nervous system or germ cell tumors; osteogenic sarcoma, and as preparation for a stem cell or bone marrow transplant). It has vastly reduced side effects compared to cisplatin. Cisplatin and carboplatin belong to the group of platinum-based antineoplastic agents, and interact with DNA to interfere with DNA repair.

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Bleomycinused vs skin cancer(see slides 12-13)

N N

NH

NH2CONH2

NH2

H2N

CH3 HN

OO HO

CH3

O

HN

NH N S

N

O

ROO

DRUGS ACTING ON DNA Chain cutters

BLEOMYCIN A2 R = NHCH2CH2CH2SMe2

BLEOMYCIN B2 R = NHCH2CH2CH2CH2NHC(NH2)=NH

CH3 HNNH

CH3

OCH3HO S

O

ONH

N

O

HO

OH

OH

OH

OH

OH

O

O NH2

Bleomycin is mostly used to treat cancer. This includes Hodgkin's disease, non-Hodgkin's disease, testicular cancer, ovarian cancer, and cervical cancer. It can be given intravenously, by injection into a muscle or under the skin. It may also be


• Intercalating agent (shown earlier)• Abstracts H from DNA to generate radicals using oxidized iron complex• Radicals react with oxygen resulting in chain cutting• Bleomycin also inhibits repair enzymes

41

muscle or under the skin. It may also be administered inside the chest to help prevent the recurrence of a fluid around the lung due to cancer

• Generates DNA diradical• DNA diradical reacts with oxygen• Results in chain cutting

Chain cutters

O

OMe

I

CH3

O

OMe

S

O

OH3C

OH

O NH

OH3C

HOO

H

SS

SH3C

HOO

NHCO2Me

Calicheamicin 1I

Antitumour agentEnediyne

O

OHMeOHO

H3CO

MeO

HN

H3C

ed y esystem

The calicheamicins are a class of enediyne antitumor antibiotics derived from the bacterium Micromonospora echinospora, with calicheamicin g1 being the most notable. It was isolated originally in the mid-1980s from the chalky soil, or "calichi pits", located in Kerrville, Texas. It is extremely toxic to all cells . N-acetyl dimethyl hydrazide calicheamicin was developed and marketed as targeted therapy against the non-solid tumor cancer acute myeloid leukemia (AML). Calicheamicin 1 and the related enediyne esperamicin are the two of the most potent antitumor agents known.


D

D

H

H

?

H

H

D

D

proposeddiradical

intermediate

H

H

D

D

Bergman reaction (1972)

D

D

H

H

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22


HO OHO

O

Michael HOO

Chain cutters

Mechanism

H

RS

NHCO2Me

SMeS

Nu

RS

NHCO2Me

HOO

HO

O

Michaeladdition

RS

NHCO2Me


Cycloaromatisation

R

S

HONHCO2Me

O2

Oxidativecleavage

DNA DNA(Diradical)

RS

HO

H

H

NHCO2Me

44

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Neocarzinostatin is a macromolecular chromoprotein enediyne antitumor antibiotic secreted by Streptomyces macromomyceticus. It consists of two parts, a labile chromophore (shown) and a 113 amino acid protein to which the chromophore is tightly and non-covalently bound with high affinity (Kd ~ 10-10 M). The non-protein component is a very potentnon protein component is a very potent DNA-damaging agent; However it is extremely unstable and the role of the protein is to protect it and release it to the target DNA. Opening of the epoxide under reductive conditions present in cells creates favorable conditions for a Bergman cyclization, that leads to formation of benzyne, followed by DNA strand cleavage. As a medicine it is among the most potent


known, and in Japan it is the only analog used clinically (against liver cancer). Neocarzinostatin likely evolved to kill bacteria that compete with the producing organism. Because it achieves this by causing DNA damage, however, it is capable of harming tumor cells, as well and was developed for its anticancer properties.

Azidothymidine (AZT) (Zidovudine;Retrovir) HN

N

O

CH3

O

OHO

HN

N

O

CH3

O

OOP

O

OH

OP

O

OH

OP

O

OH

HO

DRUGS ACTING ON DNA Chain terminators

HN

N

O

CH3

O

OOP

O

OH

OP

O

OH

OP

O

OH

HO

• Azidothymidine is a prodrug used in the treatment of HIV• AZT is phosphorylated to a triphosphate in the body• Triphosphate has two mechanisms of action

- inhibits a viral enzyme (reverse transcriptase)- is added to growing DNA chain and acts as chain terminator

N3

N3

Chain terminating group

N3


Azidothymidine (AZT) reduces the replication of the virus and leads to improvements in both symptoms and blood tests. It can also be used to prevent HIV transmission, such as from mother to child during the period of birth or after a needle stick injury. Used by itself in HIV-infected patients, AZT slows HIV replication in patients, but does not stop it entirely. HIV may become AZT-resistant over time, and therefore AZT is now usually used in conjunction with other anti-HIV drugs in the combination therapy called highly active antiretroviral therapy (HAART = highly active antiretroviral therapy).

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O

O

PO O

N

NH

O

O

N

N N

N

O P O

O

OHH2N

O

O

P OO

O

PO O

O

NTP

N

HN

O P O

N

O

O

O

P OO

O

PO O

O

O

N

N

Chain terminating

group

Azidothymidine (AZT)

O

O

O

PO O

O

N

N

NH2

O

N

HN N

N

O

O

P OO

OO

H2N

O

H

Usual way of

B

O P O

O

O

P O

O

O

O

P OO

O

PO O

O

Chain terminating

group

O

Aciclovir


O

P OO

Template

Growingchain

Usual way ofadding bases

ON

HN N

NO

H2N

R NN

N

NRH

H

NR R NN

N

H

BH

R NH

H B

Azides are very reactive and lose N2 easily (best leaving group in chemistry)

HN

N

O

H2N N

N

O

N

NN

N NH2

AcO

Chain terminators

Chain

HN

N

O

H2N N

N

O Chain

N

NN

N NH2

AcO

OHO

Acyclovir(Zovirax)

Famciclovir(Famvir)

OAcChainterminating group

OHO

Chainterminating group

OAc

Acyclovir, is an antiviral medication. It is primarily used for the treatment of herpes simplex virus infections, chickenpox, and hi l

Famciclovir is a guanosine analogue antiviral drug used for the treatment of various herpesvirus infections, most


Notes:• Prodrugs used as antiviral agents• Same mechanisms of action as AZT• Used vs herpes simplex and shingles

48

shingles. commonly for herpes zoster (shingles).

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Chain terminators

a) Normal replication

PG

P P

C

G

A

A

C

C

G OH3'

C

G

A

A

C

C

A

T

T

A3

DNAtemplate

Growingchain

A

T

T

A OH3'

DNA template

Growingchain

b) Chain termination

A

C

C

A

C

C


PDrug

P P

A

T

C

G

A

T

A OH3'

DNA template

Growingchain

A

T

C

G

A

T

A

Drug H3'

DNAtemplate

Growingchain

Chain termination

Mechanism of reverse transcription in class VI virus ssRNA-RT, human immunodeficiency virus (HIV). Colors mark complementary sequences (not drawn to scale). Reverse transcription occurs in the cytoplasm of host cell. In this process, viral ssRNA is transcribed by the viral reverse transcriptase protein (RT) into double stranded DNA. Reverse transcription takes place in 5' 3' direction. tRNA ("cloverleaf") hybridizes to PBS and provides -OH group for initiation of reverse transcription. Key: (U3 - promoter region),(U5 - recognition site for viral integrase); (PBS - primer binding site);(PP - polypurine section ).

1) Strong stop complementary DNA (cDNA) is formed. 2) Template in RNA:DNA hybrid is degraded by RNase H domain of reverse transcriptase 3) DNA:tRNA is transferred to the 3'-end of the template (synthesis "jumps"). 4) First strand synthesis takes place. 5) The rest of viral ssRNA is degraded by RNase H, except for PP site. 6) Synthesis of second strand of ssDNA is initiated from the 3'-end of the template. tRNA is necessary to synthesis of complementary PBS


7) tRNA is degraded 8) After another "jump", PBS from the second strand hybridizes with the complementary PBS on the first strand. 9) Synthesis of both strands is completed by the DNAP function of reverse transcriptase. Both dsDNA ends have U3-R-U5 sequences, so called long terminal repat sequences (3'LTR and 5'LTR, respectively). LTRs mediate integration of the retroviral DNA into another region of the host genome. Very error prone because no proof reading.

5/10/2017

26

DRUGS ACTING ON DNA Control of gene transcription

Notes:• Design of synthetic molecules capable of controlling gene transcription• Molecules capable of recognising and binding to

A

C

T

G

5'3'

O

O

NH

NN Me

O

N

N

H

O

Me

NMe

H

NPy

Im

Linker

g g gspecific base pairs• Hairpin polyamides containing heterocyclic rings are capable of binding to the minor groove• Binding involves amide groups and heterocycles• Particular patterns of h t li i ll

A

T

G

T

A

C

NO

H

NMe

O

ONH

N

M

N

N HO

N OMe

N H

O

H

HHp

Py

Py

Hp

ArmArm


heterocyclic rings allow recognition of perticular base pairs• Capable of inhibiting transcription• Designed to bind to regulatory element of a gene

51

T AMe

ON

H

N

O

H

NMe

MeH

NN

Me

N

Me

O

N

HPy

Im

DRUGS ACTING ON rRNA

Antibiotics – attack bacteria

O2N

CH2OH

HO H

Me

O

HNMe

OHOH

H2N C

NH

NH

O

HN C

NH

NH2

O

H

OHH HO

HH

OHH

H

CHO

H

CH2OH

HN H

CO CHCl2

Chloramphenicol(vs typhoid)

RO

Me

OH

O

OH

O

MeO

O

C

Me Me

OMe O MeMe

OH

H

H

H

H

H

H

H

H

Rifamycins

StreptomycinO

O

CHO

OHMe

CH2OH

H

HO

H

OH

H

H

MeHN

H

H

O

CH3NMe2

HO


Rifamycins

OOHOOH

Cl

OH

NMe2

O

HO

NH2

Me H

OH

Chlortetracycline(Aureomycin)

HO

O

O

H3C

CH3

CH3

O

CH3HO

H3C

OO

O

CH3

CH3

MeOH

OMeErythromycin 52

5/10/2017

27

O2N

CH2OH

HO

HN

H

H

CO CHCl2

Chloramphenicol(vs typhoid)

Chloramphenicol is an antibiotic useful for the treatment of a number of bacterial infections. This includes as an eye


ointment to treat conjunctivitis. By mouth or by injection into a vein it is used to treat meningitis, plague, cholera, and typhoid fever (largely drug resistant now). Monitoring of blood levels of the medication and blood cell levels every two days is recommended during treatment. Common side effects include bone marrow suppression, nausea, and diarrhea. The bone marrow suppression may result in death. To reduce the risk of side effects treatment duration should be as short as possible. It has excellent blood-brain barrier penetration. Chloramphenicol is active against the three main bacterial causes of meningitis: Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Chloramphenicol is extremely lipid-soluble; it remains relatively unbound to protein and is a small molecule. It penetrates effectively into all tissues of the body, including the brain. Chloramphenicol increases the absorption of iron.

R

Me

O

HNMe

Me

OH

O

OHOH

OH

O

MeO

Me Me

H

HHH

The rifamycins are a group of antibiotics that are synthesized either naturally by the bacterium Amycolatopsis rifamycinica or artificially. They are a subclass of the larger family of ansamycins. Rifamycins are particularly effective against mycobacteria, and are therefore used to treat tuberculosis, leprosy, and mycobacterium avium complex (MAC) infections.

Isoniazid is a prodrug and must be activated by a bacterial catalase-peroxidase enzyme in Mycobacterium tuberculosis called KatG, which catalyzes the formation of the isonicotinic acyl radical, which spontaneously couples with NADH to form the nicotinoyl NAD adduct This complex binds

O

COMe O Me

MeOH

H

HHH

Rifamycins

couples with NADH to form the nicotinoyl-NAD adduct. This complex binds tightly to the enoyl-acyl carrier protein reductase known as InhA, thereby blocking the natural enoyl-AcpM substrate and the action of fatty acid synthase. This process inhibits the synthesis of mycolic acids, which are required components of the mycobacterial cell wall. A range of radicals are produced by KatG activation of isoniazid, including nitric oxide,

Rifamycins have been used for the treatment of many diseases, the most important one being HIV-related tuberculosis.

OHN H


N

O NNH2

bacterial catalase-peroxidase enzyme

N

O

Forma a complex binds to protein important in mycolic fatty acid metabolism and blocks fatty acids used in cell wall of terberculosis bacteria.

Isoniazid

N R

N

O N RH

Fe

O

+4

N

O N R

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28

St t i

H2N C

NH

NH

O

HN C

NH

NH2

O

H

OHH HO

HH

OHH

H

CHO

OH

H

Me H

StreptomycinO

O

OH

CH2OH

H

HO

H

OH

H

H

MeHN

H

Streptomycin is an antibiotic used to treat a number of bacterial infections. This includes tuberculosis, Mycobacterium avium complex, endocarditis, brucellosis, Burkholderia infection, plague, tularemia, and rat bite fever. For active tuberculosis it is often given together with isoniazid, rifampicin, and pyrazinamide. Common side effects include feeling like the world is spinning, vomiting, numbness of the face, fever, and rash.


Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells through mechanisms that are still not understood. Speculation on this mechanism indicates that the binding of the molecule to the 30S subunit interferes with 50S subunit association with the mRNA strand. This results in an unstable ribosomal-mRNA complex, leading to a frameshift mutation and defective protein synthesis; leading to cell death. Humans have ribosomes which are structurally different from those in bacteria, so the drug does not have this effect in human cells. Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-negative bacteria, and is therefore a useful broad-spectrum antibiotic.

OOHOOH

Cl

OH

NMe2

O

HO

NH2

Me H

OH

Chlortetracycline (trade name Aureomycin, Lederle) is a tetracycline antibiotic, the first tetracycline to be identified. It was discovered in 1945. Duggar identified the antibiotic as the product of an actinomycete In veterinary medicine, chlortetracycline is commonly used to treat conjunctivitis in cats and other animals. Chlortetracycline may increase the anticoagulant activities The risk or severity of adverse effects can be2

Chlortetracycline(Aureomycin)

activities . The risk or severity of adverse effects can be increased when chlortetracycline is combined with acitretin, adapalene, or alitretinoin.


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HO

O

O

H3C

CH3

CH3

O

CH3

CH3

HO

H3C

OO

O

CH3

CH3

NMe2

HOErythromycin is an antibiotic useful for the treatment of a number of bacterial infections, including respiratory tract infections, skin infections, chlamydia infections, pelvic inflammatory disease, and syphilis. It may also be used during pregnancy to prevent Group B streptococcal infection in the newborn. An eye ointment is routinely recommended after delivery to prevent eye infections in the newborn. Common side effects include abdominal cramps, vomiting, and

OMe

OH

OMeErythromycin

p , g,diarrhea. More serious side effects may include Clostridium difficile colitis, liver problems, prolonged QT, and allergic reactions. It is generally safe in those who are allergic to penicillin

Erythromycin displays bacteriostatic activity or inhibits growth of bacteria. By binding to the 50s subunit of the bacterial rRNA complex, Erythromycin interferes with aminoacyl translocation, preventing the transfer of the tRNA


bound at the A site of the rRNA complex to the P site of the rRNA complex. Without this translocation, the A site remains occupied, thus the addition of an incoming tRNA and its attached amino acid to the nascent polypeptide chain is inhibited. This interferes with the production of functionally useful proteins, which is the basis of this antimicrobial action.

Protein synthesis

DRUGS ACTING ON mRNA Antisense Therapy

m-RNA

antisensemolecule

U C A G A U G C A A

A G U C U A C G U U

AAUG A A G Um-RNA

antisensemolecule


Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off". This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an "anti-sense" oligonucleotide (ASO) because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the "sense" sequence (so that a sense segment of mRNA " 5'-AAGGUC-3' " would be blocked by the anti-sense mRNA segment " 3'-UUCCAG-5' ").

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Advantages

• Same effect as an enzyme inhibitor or receptor antagonist

Antisense Therapy

DRUGS ACTING ON mRNA

• Same effect as an enzyme inhibitor or receptor antagonist• Highly specific where the oligonucleotide is 17 nucleotides or more• Smaller dose levels required compared to inhibitors or antagonists• Potentially less side effects

Disadvantages

• ‘Exposed’ sections of mRNA must be targeted


p g• Instability and polarity of oligonucleotides (pharmacokinetics)• Short lifetime of oligonucleotides and poor absorption across cell membranes

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• Short segments of double stranded RNA• Recognised by enzyme complex RISC to produce single stranded RNA – smallinterfering or small inhibitory RNA (siRNA)

• Binds to complementary region of mRNA• mRNA is cleaved by enzyme complex

DRUGS ACTING ON mRNA Micro-RNA (miRNA)

y y p

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssRNA) fragment, such as microRNA (miRNA), or double stranded small interfering RNA (siRNA). The single strand acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene silencing and defense against viral infections.


RISC

miRNA

RISC

miRNA

RISC

siRNA

RISC

mRNA

60

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Advantages• siRNAs have potential to be used in gene therapy• Greater efficiency in silencing mRNA than conventionalantisense therapy

Micro-RNA (miRNA)

antisense therapy• One siRNA could lead to cleavage of several mRNA

molecules

Problems• siRNAs need to be metabolically stable


siRNAs need to be metabolically stable • Need to reach target cells• Need to enter target cells

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Epigenetics – a new frontier: DNA methylation, methylcytosine, histone acylation


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Five major families of histones exist: H1/H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 are known as the core histones, while Histones H1 and H5 are known as the linker histones.

Histones make five types of interactions with DNA:

1. Helix-dipoles form alpha-helixes in H2B, H3, and H4 cause a net positive charge to accumulate at the point of interaction with negatively charged phosphate groups on DNA2 H d b d b t th DNA b kb d th id th i h i f hi t t i2. Hydrogen bonds between the DNA backbone and the amide group on the main chain of histone proteins3. Nonpolar interactions between the histone and deoxyribose sugars on DNA4. Salt bridges and hydrogen bonds between side chains of basic amino acids (especially lysine and arginine) and phosphate oxygens on DNA5. Non-specific minor groove insertions of the H3 and H2B N-terminal tails into two minor grooves each on the DNA molecule

Histones are highly basic facilitating DNA-histone interactions and contribute to their water solubility.

Histones are subject to post translational modification by enzymes primarily on their N-terminal tails, but


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Histones are subject to post translational modification by enzymes primarily on their N terminal tails, but also in their globular domains. Such modifications include methylation, citrullination, acetylation, phosphorylation, SUMOylation, ubiquitination, and ADP-ribosylation. This affects their function of gene regulation.

In general, genes that are active have less bound histone, while inactive genes are highly associated with histones during interphase. Histones are evolutionarily conserved, and any deleterious mutations appear to be severely maladaptive. All histones have a highly positively charged N-terminus with many lysine and arginine residues.

Methylations in biochemistryCH3

NHNH2N N

HNH2N

1 8

Me-vit B12

NN

NO

Ar

H

H

HN

N

NO

Ar

CH3

H

H

N5 methyl-THF

12

3 4 5 6

78

9

10

5

Me-vit B-12 vit B-12

S S

CH3H

ATP

S

NH

CH3

ON

N

N

H2N

THF(tetrahydrofolate)


NH3

HO2C

homocysteine

NH3

HO2C

methionine S-adenosyl methionine (SAM)

NH3

HO2C

N

NH3C

NH2

O

DNA

5-Methylcytosine

OHOH

N

SAM

N

NH

NH2

O

DNA

cytosine

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Citrullination or deimination is the conversion of the amino acid arginine in a protein into the amino acid citrulline. Enzymes called peptidylarginine deiminases (PADs) replace the primary ketimine group (=NH) by a ketone group (=O).

Citrullination controls the expression of genes, particularly in the developing embryo. The immune system often attacks citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Citrulline is not one of the 20 standard amino acids encoded by DNA in the genetic code. Instead, it is the result of a post-translational modification.

Citrullination is distinct from the formation of the free amino acid citrulline as part of the urea cycle.

Arginine is positively charged at a neutral pH, whereas citrulline is uncharged. This increases the hydrophobicity of the protein, leading to changes in protein folding. Therefore, citrullination can change the structure and function of proteins.

Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone proteins, while other proteins, like fibrin and vimentin, can get citrullinated during cell-death and tissue inflammation.

Fibrin and fibrinogen may be favored sites for arginine deimination within rheumatoid joints. Test for presence of anti-citrullinated protein (ACP) antibodies are highly specific (88-96%) for rheumatoid arthritis (RA), about as sensitive as rheumatoid factor (70-78%) for diagnosis of RA, and are detectable from even before the onset of clinical disease.

H2N NH2 H2N OH

B H2NNH2

O

H


NH

N

O

N

H

Harginine

+NH

N

O

N

H

Hcitrulline

+ NH3PAD(enzyme)

OH

NH

N

O

N

H

H

O

H

etc.

Acylations in biochemistry

Histone proteins are acetylated and deacetylated on lysine residues in the N-terminal tail as part of gene regulation. The regulation of transcription factors, effector proteins, molecular chaperones, and cytoskeletal proteins by acetylation and deacetylation is a significant post-translational regulatory mechanism. These regulatory mechanisms are analogous to phosphorylation and dephosphorylation by the action of kinases and phosphatases. Not only can the acetylation state of a protein modify its activity but there has been recent suggestion that this post-translational modification may also crosstalk with phosphorylation, methylation, ubiquitination, sumoylation, and others for dynamic control of cellular signalingcellular signaling.

N

O

N

H

H

HNS

O

Co-A

H

O

lysine

N

O

N

H

H

N O

lysine

SCo-A

B H

N

O

N

H

H

NH

lysine

Co-A-SH


O Glu-Enzacetyl Co-A

glutamate aa

H

HO Glu-Enz

glutamate aa

OO

acylatedlysine

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Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination In contrast to ubiquitin SUMO is not used

Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues (ubiquitously) of eukaryotic organisms. There are four genes in the human genome that produce ubiquitin: UBB, UBC, UBA52 and RPS27A. Ubiquitination can affect proteins in many ways: it can signal for their degradation via the proteasome alter their cellular location affect their activity


ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.

proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions. Ubiquitination is carried out in three main steps: activation, conjugation, and ligation, performed by ubiquitin

ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein. It is a reversiblepost-translational modification that is involved in many cellular processes, including cell signaling, DNA repair, gene regulation and apoptosis. Improper ADP-ribosylation has been implicated in some forms of cancer. It is also the basis for the toxicity of bacterial compounds such as cholera toxin, diphtheria toxin, and others.

O

N

H2N

OO ADP

H2N

ON O P O P O

ON

O

O

O

O

H2N N

N

N

OH OH OH

OH

NAD+

NH

H

NH

Arg

O

O Glu

OO ADPN

H

H2N

NH

Arg OH

OH

OH


O

OH

O Glu

OH

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0 Med Chem, chapter 9.ppt - Cal Poly Pomonapsbeauchamp/pdf/499_Med_Chem_topic... · 2017-05-10 · Mechanism of action • Contain planar aromatic or heteroaromatic ring systems