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Tirapazamine: an Anticancer
Pro-drug with Antimicrobial
Activity
Zarnaben (Zarna) Shah
Department of Biochemistry
McGill University, Montreal
April 2012
A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Masters of Science
© Copyright by Zarna Shah (2012)
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To my parents...
Anil & Harsha Shah
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ABSTRACT
Antibiotics are one of the most successful chemotherapeutic agents in the
history of mankind. They have proven their necessity from “from cradle to
grave” and played a central role in global socioeconomic development for past
seven decades. However, rapidly increasing bacterial resistance to existing
therapy has caused an urgent need for development of new antibiotics.
Tirapazamine (TPZ, 3-amino-1,2,4-benzotirazine 1,4 dioxide) is a pro-drug under
phase III clinical trials for various types of cancers with unique mechanism of
action. In the absence of oxygen, TPZ forms a toxic radical species with the help
of cellular enzymes which ultimately causes DNA damage. Since TPZ has been
successfully tested both in animal cells and yeast, we were curious to learn about
its antibiotic potential. Our results indicate that fluoroquinolone resistant E. coli,
Methicillin-resistant Staphylococcus aureus and C. difficile strains are sensitive
to TPZ at three orders of magnitude lower concentration than what has been
used in cancer treatments. Time killing experiments in E. coli provide preliminary
evidence for bactericidal nature of TPZ and its involvement in DNA damage is
indicated by hypersensitivity of mutants deficient in homologous recombination.
In animal cells, TPZ is activated by conversion of the pro-drug by reductases.
Similarly, E. coli mutants of argC, yeiA and ydhV are resistant to TPZ suggesting
putative reductases produced by these loci are involved in metabolizing TPZ into
a toxic compound. Pathogenic strains of C. difficile are sensitive to TPZ starting
from 7.5ng/ml which is lower than other antibiotic concentrations previously
tested. While further experiments are required to decipher the detail
mechanism by which TPZ operates, TPZ and its derivatives might be a promising
alternative for treating pathogens that are resistant to existing antibiotics.
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RÉSUMÉ
Les antibiotiques sont un des agents thérapeutiques les plus couronnés de
succès dans l’histoire de l’humanité. Ils sont d’une très grande utilité tout au
cours de la vie et ils ont joué un rôle primordial pour le développement socio-
économique lors des sept dernières décennies. Cependant, une croissance
rapide de la résistance bactérienne aux thérapies courantes a créé un urgent
besoin pour le développement de nouveaux antibiotiques. La tirapazamine (TPZ,
3-amino-1,2,4-benzotirazine 1,4 dioxide) est une prodrogue utilisée pour le
traitement du cancer. La TPZ est maintenant testée dans des essais cliniques en
phase III. La TPZ a un mécanisme d’action unique. En absence d’oxygène et grâce
à l’action d’enzymes cellulaires, la TPZ forme un radical toxique qui cause des
dommages à l’ADN. Etant donné que la TPZ a été testée avec succès dans des
cellules animales et chez la levure, nous étions intéressés de tester la TPZ
comme antibiotique potentiel. Nos résultats indiquent que des souches de E. coli
résistantes à la fluoroquinoline, des souches de Staphyllocus aureus résistantes à
la méthycilline ainsi que des souches de C. difficile sont sensibles à la TPZ à des
concentrations mille fois inférieures à celles utilisées pour le traitement de
cancers. Des expériences de mortalité chez E. coli suggérent que la TPZ est
bactériocide. De plus, l’hypersensibilité à la TPZ de mutants de E. coli déficients
pour la recombinaison homologue suggère que cette drogue produit des
dommages à l’ADN. Dans les cellules animales, la prodrogue TPZ est activée par
la conversion en un produit toxique via l’action de réductases. De façon similaire,
des mutants de E. coli comportant des délétions pour les gènes argC, yeiA
ou ydhVsont résistants à la TPZ. Ceci suggère que les réductases potentielles
encodées par ces gènes sont impliquées dans la conversion de la TPZ en un
composé toxique. Ceci suggère que ces réductases sont impliquées dans la
conversion de la TPZ en un produit toxique. Fait intéressant, des souches
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pathogènes de C. difficile sont sensibles à la TPZ et ce, à des concentrations aussi
basses que 7.5 ng/µl, une concentration inférieure aux autres antibiotiques
testés précédemment. Des expériences additionnelles seront requises afin de
mieux comprendre le mode d’action de la TPZ. La TPZ et ses dérivés peuvent être
une alternative prometteuse pour traiter des infections par des bactéries
résistantes aux antibiotiques couramment utilisés.
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ACKNOWLEDGEMENT
This project would not have been possible without support of numerous generous contributors. First and foremost, I would like to thank my supervisor Dr. Bernard Turcotte for honoring me with this great project and mentoring my development as a scientist. I would like to thank my committee members Dr. Albert Berghuis and Dr. James Coulton for their meaningful insights and constructive criticism. We are grateful to Dr. Vivian Loo and Susan Fenn at the Montreal General Hospital for providing their expertise and facility for C. difficile related experiments. Dr. Marc Drolet’s lab and Valentine Usongo at the Université de Montréal assisted me with in vivo gyrase assay and Dr. Karine Auclair’s lab generously supplied purified cytochrome p450 reductase enzyme for in vitro gyrase assay. Furthermore, I am thankful to Sanofi-synthelabo for providing Tirapazamine powder, Media lab at McGill for Brain-heart infusion broth and leaked sheep blood, Dr. Anderson from Université Laval for Salmonella strains TA100 and TA98 for Ames test, Keio collection (Japan) for providing E. coli reductase mutant strains, Dr. Robert Kerns from University of Iowa for providing gyrase mutant strains, and Dr. Brigitte Lefebvre from Laboratoire de santé publique du Québec for S. aureus strains. The project was funded by CIHR and initial work was done by an undergraduate student in our lab, Raya Mahbuba. Moreover, I want to thank countless other researchers, whose work I have read and referenced throughout my graduate studies for their findings have provided the foundation for this project to materialize. Ultimately, the intricate task of editing my thesis (formatting and grammatical errors) was patiently executed by: Dr. Bernard Turcotte (Associate professor at McGill University), Rukshan Mehta (Masters in Social Work from University of Toronto) and Nirmal Shah (Environmental Engineering student at University of Guelph). Finally, Dr. Greg Marczynski (Associate professor at McGill University) provided an invaluable perspective as an external examiner of this thesis.
DEEP GRATITUDE
I want to express my utmost gratitude to God for giving me smruti (memory), shakti (strength), and shanti (peace). I am immensely grateful to my parents, my brother, and my extended family for their love and blessings without which I would not have been able to pursue my studies. I would like to express my gratitude to my present and past teachers, mentors, professors, and Turcotte lab members (Karen, Nadya and Natalia) for their valuable contribution in my wholesome development. A grand gesture of appreciation to my friends: Poonamben, Jeegarbhai, Amenda, Vesna, Alessandra, and Shuvadeep for their emotional support during my time at Montréal. I like to thank Montréal for having excellent public transportation, best bread and cheese I ever had, and the world-class hot chocolate of juliette et chocolat. Last but by no means least, I will be forever in debt to McGill University for adding my name to a highly honorable group of alumni and making me part of a glorious scientific heritage.
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TABLE OF CONTENTS
ABSTRACT .............................................................................. i
RÉSUMÉ ................................................................................. ii
ACKNOWLEDGEMENT ............................................................ iv
TABLE OF CONTENTS ............................................................. v
LIST OF ABBREVIATIONS .................................................... viii
THESIS OUTLINE & RATIONALE ............................................. 1
I. LITERATURE REVIEW.......................................................... 2
I.1 Antibiotics ........................................................................ 2
I.1.1 Urgent need for new classes of antibacterial drugs .......................... 2
I.1.2 Antibiotics: the age old battle with bacteria ..................................... 3
I.1.2.1 Origin and historic timeline ................................................................... 3
I.1.2.2 Antibiotics– agents against life............................................................... 4
I.1.2.3 Antibiotic classification ......................................................................... 5
I.1.2.3.1 Natural vs. synthetic .......................................................................... 5
I.1.2.3.2 Classification based on bactericidal and bacteriostatic activity ............ 5
I.1.2.3.3 Classification based on mechanism of action ...................................... 6
I.1.2.3.3.1 Summary of cell wall biosynthesis as a target .................................. 6
I.1.2.3.3.1.1 Cellular mechanism of vancomycin ............................................... 6
I.1.2.3.3.2 Protein biosynthesis as a target ....................................................... 7
I.1.2.3.3.3 Summary of DNA replication/repair mechanism .............................. 7
I.1.2.3.3.3.1 Cellular mechanism of norfloxacin ................................................ 7
I.1.3 Drug production .............................................................................. 8
I.1.3.1 Antibiotics in nature ............................................................................. 8
I.1.3.1.1 Why do microbes make antibiotics? .................................................... 8
I.1.3.1.2 Protection of antibiotic producers ....................................................... 9
I.1.3.2 Synthetic production ............................................................................. 9
I.1.3.2.1 Genomic based approach ................................................................. 10
I.1.4 Antibiotic resistance ...................................................................... 10
I.1.4.1 Resistance is here to stay .................................................................... 10
I.1.4.2 Intrinsic Resistance ............................................................................. 11
I.1.4.3 Molecular epidemiology of acquired resistance ..................................... 11
I.1.4.4 Cellular modes of acquiring resistance ................................................. 13
I.1.4.5 Combating antibiotic resistance........................................................... 14
I.1.5. Light at the end of the tunnel ....................................................... 15
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I.2 Tirapazamine ................................................................. 16
I.2.1 Origin of the pro-drug.................................................................... 16
I.2.2 Chemical structure and properties ................................................ 18
I.2.3 Mechanism of preferential toxicity ................................................. 18
I.2.3.1 Pro-drug activation by reducatases ...................................................... 19
I.2.3.2 Oxidative damage caused by TPZ ......................................................... 19
I.2.3.3 Futile cycle of enzymatic reduction under aerobic condition ................. 20
I.2.3.4 Secondary metabolites ........................................................................ 21
I.2.3.5 Macromolecules as a target for TPZ ..................................................... 21
I.2.3.5.1 Topoisomerase II .............................................................................. 22
I.2.4 TPZ as a potential anticancer drug ................................................ 22
I.2.5 Anticancer drug to serve as an antibiotic ....................................... 24
I.3 Clostridium difficile Infection......................................... 25
I.3.1 Global epidemiology of Clostridium difficile Infection (CDI) ............ 25
I.3.2 C. difficile infections are difficult to treat ....................................... 25
I.3.3 C. difficile pathogenicity ................................................................. 26
I.3.4 CDI pathology................................................................................ 27
I.3.5 Mechanism of action ..................................................................... 28
I.3.6 CDI diagnosis methods .................................................................. 29
I.3.7 CDI therapeutics ........................................................................... 30
I.3.8 Concluding remarks ...................................................................... 32
II Materials and Methods ..................................................... 33
II.1 Strains .................................................................................................... 33
II. 2 Drugs and media: ................................................................................... 35
II.3 Drug spotting assay ................................................................................. 35
II.3.1 Anaerobic Incubation ............................................................................ 35
II.4 MIC determination of gyrase mutants ....................................................... 36
II.5 C. difficile experiment ............................................................................... 37
II.6 Time-Kill assays (Killing Curves) ............................................................... 38
III. RESULTS ........................................................................ 39
III.1 TPZ has antibacterial properties .............................................................. 39
III.2 Multidrug resistant strains are sensitive to TPZ ....................................... 39
III.2.1 Fluoroquinolone resistance strains are sensitive to TPZ ........................ 39
III.2.2 MRSA strains are sensitive to TPZ ........................................................ 40
III.2.3 C. difficile strains are sensitive to TPZ................................................... 41
III.3 TPZ is bactericidal .................................................................................. 41
III.4 Mutation in HR genes results in TPZ hypersensitivity .............................. 42
III.5 Bacterial reductase mutants are resistant to TPZ .................................... 42
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IV. DISCUSSION .................................................................... 44
IV.1 TPZ as a potential antibiotic ........................................................... 44
IV.1.1 C. difficile is sensitive to TPZ ................................................................ 44
IV.2 Mechanism of action of TPZ in bacteria .......................................... 45
IV.3 Hypoxia dependence of TPZ ............................................................ 47
IV.4 Resistance to TPZ ........................................................................... 48
IV.4.1 Reductase mutants resistant to TPZ ..................................................... 49
V. FUTURE DIRECTIONS ....................................................... 50
V.1 Conclusion ...................................................................................... 52
VI. FIGURES ......................................................................... 53
Figure 1.1: The perfect storm ................................................................ 53
Figure 1.2: Drying pipeline of antibiotics. .............................................. 53
Figure 1.3: Structural formula of various antibiotics ............................. 54
Figure 1.4: Logarithmic growth curve of bacteria ................................... 55
Figure 1.5: Drug resistance mechanisms ............................................... 55
Figure 1.6: Chemical structure of Tirapazamine .................................... 56
Figure 1.7: General mechanism of preferential cytotoxicity of TPZ ......... 56
Figure 1.8: A schematic of DNA gyrase mechanism. .............................. 57
Figure 1.9: C. difficile under microscope ................................................ 57
Figure 3.1: Wild-type E. coli sensitivity to TPZ ....................................... 58
Figure 3. 2: Fluoroquinolone resistant strains are sensitive to TPZ ........ 59
Figure 3.3: S. aureus strains are sensitive to TPZ under low O2 levels. .. 60
Figure 3.4: C. difficile is sensitive to TPZ. ............................................... 61
Figure 3.5: TPZ is bactericidal . ............................................................. 62
Figure 3.6: TPZ is might be involved in direct or indirect DNA damage. . 63
VII. TABLES.......................................................................... 65
Table 1.1 ............................................................................................... 65
Table 1.2 ............................................................................................... 66
Table 3.1 ............................................................................................... 67
Table 5.1 ............................................................................................... 68
VIII. BIBLIOGRAPHY ............................................................. 71
IX. APPENDIX ....................................................................... 88
Appendix A ............................................................................................ 88
Appendix A2 .......................................................................................... 90
Appendix B ............................................................................................ 91
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LIST OF ABBREVIATIONS
BLAST Basic Local Alignment Search Tool
BTZ Benzotriazine or benzotriazinyl
CDI Clostridium difficile infection
CFU Colony forming units
CLSI Clinical and Laboratory Standards Institute
DNA Deoxyribonucleic Acid
DSB Double stranded breaks (of DNA)
E. coli Escherichia coli
EIA Enzyme immuno assay
EMS Ethyl methanesulfonate
FDA Food and Drug Administration (US)
FQRP Fluoroquinolone-resistant Pseudomonas aeruginosa
GDH Glutamine dehydrogenase
GTPases Guanosine triphosphate hydrolases
HCR Hypoxic cytotoxicity ratio
HIF Hypoxia inducible factor
HNC Head and neck cancer
HR Homologous recombination
IBD Inflammatory bowel disease
IDSA Infectious Disease Society of America
LPS Lipopolysaccharide
MDR Multi-drug resistant
MIC Minimum inhibitory concentration
MRSA Methicillin-resistant Staphylococcus aureus
MTD Maximum tolerated dose
NSCLC Non-small cell lung cancer
ORFs Open reading frames
PG Peptidoglycan
PMC Pseudomembranous colitis
PPI Proton pump inhibitor
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QRDR Quinolone resistance determining region
ROS Reactive Oxygen Species
RT-PCR Real-time polymerase chain reaction
TPZ Tirapazamine
VRE Vancomycin-resistant Enterococcus
WBC White blood cells
WHO World health organization
WT Wild type
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THESIS OUTLINE & RATIONALE
Bacterial resistance is a socioeconomic burden as well as a hazardous
threat to public health. Multidrug resistant (MDR) bacteria such as Clostridium
difficile (C. difficile), Vancomycin-resistant Enterococcus (VRE) and Methicillin-
resistant Staphylococcus aureus (MRSA) are responsible for 25,000 deaths per
year in the European Union alone [1]. Bacterial resistance is also one of the
reasons large pharmaceutical companies backing out their investment in
antibiotic research. With 20,000 deaths due to C. difficile infection in USA
annually [2], it is up to the academia and small pharmaceutical companies to
develop solutions against these pathogens. There is an urgent global need for
new classes of antibiotics with new mechanism of action. Tirapazamine (TPZ) is
an anticancer pro-drug with hypoxia specific activity against tumor cells. It is
currently in phase III clinical trials for non-small cell lung cancer (NSCLC), head
and neck cancer (HNC) and cervical cancer. The preferentially toxic mechanism
of TPZ action in eukaryotes generates active radical species which ultimately
causes DNA damage. The toxicity of TPZ is inversely proportional to oxygen
availability in the cell. Exploiting this unique mechanism for MDR pathogens can
greatly improve our capacity to control resistance. The purpose of this research
was to explore the potential of TPZ as an antibacterial drug and obtain basic
mechanistic insights of TPZ in prokaryotes. I found that fluoroquinolone resistant
E. coli, MRSA and C. difficile strains are sensitive to TPZ at 1000 times lower
concentration than what is used for cancer treatment. Also, I identified a group
of putative bacterial reductases involved in converting TPZ from a pro-drug to an
active species. Furthermore, I observed bactericidal nature of TPZ and gathered
evidences that suggest TPZ mediated DNA damage. All results are outlined,
presented and interpreted by means of this thesis. Their limitations and future
experiments are also discussed at the end of the thesis.
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I. LITERATURE REVIEW
I.1 Antibiotics
Definition: Antibiotics are chemical compounds that prevent growth of microbes, namely, bacteria and fungi. However, due to the large proportionate volume and diversity of therapeutic antibacterial available in the market, the word “antibiotic” is often used in reference to “antibacterial.” For the purpose of this thesis the terms ‘antibiotic’ and ‘antibacterial’ will be used interchangeably.
I.1.1 Urgent need for new classes of antibacterial drugs
Since the discovery of β-lactams in 1930s, antibiotics have been used by
individuals during numerous life threatening situations proving their necessity
“from cradle to grave” [3]. Antibiotics have been front and center of modern
society’s fight against infections for the past seven decades and have globally
played pivotal role in economic and social advancement. Today, pharmaceutical
industries and health related research are major part of developed world
economies. Global pharmaceutical market was estimated to be more than US
$800 billion in 2009. Anti-infective agents consist 5% of the global
pharmaceutical market [4, 5]. Despite the raising industrial interest in antiviral
and vaccines; antibiotics have shown an average annual growth of 4% from 2004
to 2009 [5].
Shortage of antibacterial drugs is one of the biggest threats to public
health in the 21st century. Increasing cases of MDR strains such as MRSA, VRE
and Fluoroquinolone-resistant Pseudomonas aeruginosa (FQRP) combined with
declining interest of pharmaceutical companies in antibacterial research creates
a recipe for “a perfect storm” [Figure 1.1] [6]. Only three new classes of drugs
have been introduced since 1970, and European Medicines Agency warns of a
drying pipeline for new drugs [1, 7] [Figure 1.2]. Furthermore, there is a growing
fear of “post-antibiotic” era due to fast emerging resistance in pathogenic
bacteria. According to a 2009 report, MDR bacteria are responsible for 25,000
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deaths per year in the European Union alone [1]. MDR tuberculosis is a growing
health concern with nearly 450,000 new cases registered every year, and 33% of
these prove to be lethal [8]. Emerging drug resistance is also the core reason
large companies are withdrawing their investment from development of
antibacterial drugs. It takes an investment of $800 million to $2 billion in the
span of few decades to develop a new drug [9]. Phase III clinical trials for a single
drug on a large group of patients can cost up to US $70 million. As soon as
resistance develops, the antibiotic loses its market. Therefore, a large initial
investment in antibiotics is a risk prone undertaking. Moreover, resistance is an
economic burden on the health care system. There is an estimated loss of $21-
$34 billion annually in the United States alone due to resistant strains [10].
Compared to diabetes, neurological drugs, and anti-cancer drugs;
antibiotics, being a curative treatment with short prescription duration, are
much less profitable for the pharmaceutical industry. New antibiotics face a large
competition from generic brands and are often saved as a last resort, limiting
their sales for resistant pathogens only. Furthermore, increasingly strict
regulatory policies make the approval process for new drugs lengthy, risky and
expensive [11]. Today, a new drug must be proven “superior” to all existing
drugs in order to be approved as oppose to “non-inferior”. Lack of proper
guidelines towards clinical trials and accepted model to prove safety and efficacy
of the drug makes the process highly ambiguous [12]. As a result, large
pharmaceutical companies are rapidly withdrawing their investment from
antibacterial drug development.
I.1.2 Antibiotics: the age old battle with bacteria
I.1.2.1 Origin and historic timeline
Antibiotics are one of the most successful chemotherapeutic agents in
the history of mankind which actually cure the disease as opposed to curing the
symptoms or simply delaying the disease development. In 1928, Alexander
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Fleming observed a contamination on the plate which was preventing the
growth of Staphylococci bacteria [13]. That contamination was a fungus
Penicillum notatum. Later, this fungus was isolated and its secretion was purified
to be named ‘penicillin’ [Figure 1.3a]. Nevertheless, it would be another decade
and a half before penicillin’s true potential will be realized. In 1942, pioneering
researcher in antibiotics, Selman A Waksman coined the term “antibiotic” for the
microorganism product that is able to kill bacteria [14]. During his career, he
successfully identified at least 18 antibiotics including actinomycin,
streptothricin, gramicidin, and bacitracin [15]. In spite of Dr. Waksman’s
contribution, penicillin shined as the “wonder drug” at the 1946 antibiotic
conference (held by New York Academy of Science) because it acted upon strains
that were resistant to sulfonamides which was the only available antimicrobial
therapy at the time. Penicillin marked the start of the golden era of antibiotics.
I.1.2.2 Antibiotics– agents against life
Today, these “against life” agents (i.e. antibiotics), have expended their
realm beyond microorganism product; incorporating synthetic molecules made
by medicinal chemistry. Whether they are natural or synthetic, antibiotics are
designed to specifically target biochemical processes in microbes, sparing
minimal toxicity to human hosts. Antibiotics are unique class of drug molecules
because they target an organism that has short doubling time and is evolving
rapidly. Bacteria not only evolve by mutation but also by horizontal gene transfer
of resistance genes among species (see I.1.4.3). Interestingly for antibiotics, the
more they are used; less usable they become owing to a phenomenon called
“acquired resistance” (see I.1.4.4).
Today, antibiotics have found their niche beyond human medicine. An
estimated 50% of total global usage of antibiotics is for animals both as
therapeutics and as growth promoters [16]. Moreover, antibiotics are widely
used in agriculture, aquaculture, and horticulture in sub-therapeutic
concentrations. Due to the focus of this thesis on clinical usage in humans, I will
5
not address the other utilities in detail. However, the role of these alternative
usages in resistance dynamics and long term health risk of animals to human
transmittance of resistant bacteria should not be overlooked.
I.1.2.3 Antibiotic classification
I.1.2.3.1 Natural vs. synthetic
The majority of antibiotics introduced in human clinical use have been
natural products or modification of natural products. Bacteria and fungi both
produce antibiotics. Despite a huge success of naturally derived antibiotics, there
are three main classes of synthetic drugs available in the market: the sulfa drugs
(1930s), the quinolones (1960s), and the oxazolidinones (2000s). The synthetic
approach is more popular among pharmaceutical researchers since pure
compounds can be made with therapeutic specificity and utility. These two
approaches are now used simultaneously. Chemistry is used to modify the
natural compounds to increase their bioavailability and stability, broaden
spectrum activity and/or to increase their efficacy of the drug molecule against
resistant microbes [17].
I.1.2.3.2 Classification based on bactericidal and bacteriostatic activity
Another way of classification of antibiotics is based on their effect on the
bacteria. Drugs that kill the cells are termed bactericidal agents for example,
penicilins, cephalosporins, and aminoglycosides. Bacteriostatic agents are those
that prevent growth of the organism at clinical concentrations such as
chloramphenicol, erythromycin, and tetracyclins. Agents like sulfonamides can
be placed in either category depending on the cellular system or environment of
the infection. These effects are highly concentration dependent, and the same
drug at different concentrations in different environment can have altering
effect on the bacterial viability. This effect can be easily determined in vitro by
counting cell viability at different stages of bacterial growth [Figure 1.4]. Both
types of agents have their distinct purpose in the treatment. Bacteriostatic drug
6
are used so that patient’s immune system can catch up after a certain time
frame, but if the patient is immuno-compromised, it is better to prescribe
bactericidal drugs [18].
I.1.2.3.3 Classification based on mechanism of action
Antibiotics can be classified based on their economical impact, chemical
structure, the species of bacteria they act upon, and their mechanism of action.
Currently, the most adapted method of classification is based on their
mechanism of action and their target in cellular systems. There are three major
classes of antibiotics that: i) Target cell wall biosynthesis, ii) Target protein
synthesis iii) Target DNA replication/repair machinery [Table 1]. Protein synthesis
and DNA replication has enough structural differences that some components
can be inhibited selectively in prokaryotes [17]. Additionally, disruption of
bacterial membrane structure and inhibition of metabolic pathway (e.g. folic acid
biosynthesis pathways inhibited by sulfamethoxazole-trimethoprim) are minor
mechanisms of action [19]. These minor targets are unique since they are
missing their counterparts in humans. Nevertheless, cell wall biosynthesis and
protein biosynthesis on the ribosome has been the most popular targets
throughout antibiotic history.
I.1.2.3.3.1 Summary of cell wall biosynthesis as a target
Both gram-positive and gram-negative bacteria have a peptidoglycan (PG)
layer as a part of their cell wall structure. Many antibiotics in this class target
enzymes involved in PG assembly and cross linking. β-lactams, cephalosporins,
carbapenems and glycopeptide agents such as vancomycin are all examples of
antibiotics that effect cell wall biosynthesis.
I.1.2.3.3.1.1 Cellular mechanism of vancomycin
Vancomycin is widely used in the United States for serious gram-positive
infections [Figure 1.3b]. Glycopeptide class of this antibiotic is used as a last
resort particularly for MRSA and C. difficile infections [20]. Isolated from
Streptomyces orientalis in 1956, the large size of vancomycin prevents it from
7
diffusing through gram-negative cell membrane; therefore it is only effective in
gram-positive pathogens. Vancomycin targets cell wall biosynthesis by inhibiting
PG polymerase through the complex formation D-alanyl-D-alanine motif of
peptide precursor.
I.1.2.3.3.2 Protein biosynthesis as a target
Ribosome plays a key role in protein biosynthesis mechanism. Due to the
numerous steps involved in protein biosynthesis, disruption in timing or
specificity in any one of the steps can slow down growth or be lethal to bacteria.
Many antibiotics such as macrolides, linzolid, tetracycline, aminoglycosides,
streptogramins, and oxazolidinones act on different subunits of ribosome.
I.1.2.3.3.3 Summary of DNA replication/repair mechanism
DNA replication and repair are crucial to any cell survival, and disturbing
it would be the most obvious way to kill the cell. DNA gyrase (DNA type II
topoisomerase) and RNA polymerase are the two major targets of quinolone and
rifampin class of drugs, respectively. The second generation quinolones,
fluoroquinolones, are widely used due to their broad activity against both gram-
positive and gram-negative pathogens. Fluoroquinolones are synthetic versions
of naturally found compounds coumarins [Figure 1.3c] and they are marketed
under the name levofloxacin and ciprofloxacin [Figure 1.3de].
I.1.2.3.3.3.1 Cellular mechanism of norfloxacin
Norfloxacin belongs to quinolone group of drugs which targets DNA
gyrase (topoisomerase II) and topoisomerase IV [Figure 1.3f]. As a result of
quinolone binding, the DNA topoisomerase complex is stalled at the religation
step [21]. This creates double stranded breaks (DSB) in DNA, disrupts DNA
replication and eventually triggers cell death. What makes the topoisomerase a
target of interest is that it is essential, found in all bacteria, different enough
from human homolog to achieve needed specificity, and there is a possibility of
inhibition of two distinct bacterial enzymes (topoisomerase II and IV) which can
reduce the risk of resistance development [22]. Resistance to fluoroquinolones
still exists due to mutations in N-termini of both subunits of the DNA gyrase [23].
8
This region is called QRDR region of gyrA (from Ala67 to Gln106) and gyrB (from
Asp426 to Lys447) gene product [24, 25].
I.1.3 Drug production
I.1.3.1 Antibiotics in nature
The majority of antibiotics in the market have been derived from natural
sources, and only synthetically modified to increase their functional life span
against emerging resistance. According to one study, 78% of the antibiotics
marketed between 1982 and 2002 were developed from natural products [26].
Fifty-five percent commercially significant antibiotics are produced by
actinobacteria, specifically from the genus streptomycetes [27, 28]. This gram-
positive, filamentous genus triggers antibiotic production via γ-butyrolactone (A-
factor) group of auto-regulators [29]. Production of various secondary
metabolites in microbes is highly dependent on growth conditions; the same
strain can produce considerably different compounds depending on the
environment [30]. Extraction of natural products is a complex process with a
possibility of artifacts and difficulty in detection due to interference from the
presence of other molecules [31]. As a result, the interest in natural molecules
decreased in the past, until recent technological advancements have put them
back in the game.
I.1.3.1.1 Why do microbes make antibiotics?
Prima facie antibiotic production in nature should provide survival
advantage to producing organisms against limiting resources; however,
antibiotics found in the wild are often at sub-inhibitory concentrations. Also,
antibiotics are produced as secondary metabolites during the stationary phase of
bacterial growth, as opposed to at the logarithmic phase when the competition
for resources is the most fierce. Therefore, researchers have proposed that
antibiotics serve as a mode of inter-microbial communication [32]. This theory is
9
supported by the observation that A-factor group of molecules (see I.1.3.1) are
involved in quarum sensing system [33], which is a mode of inter-kingdom
signaling in bacteria [34]. In some cases, antibiotics are utilized as immune
system modulators in eukaryotes due to their anti or pro inflammatory
properties. For example, macrolides are used for their anti-inflammatory
properties in cystic fibrosis patients rather than their ability to defeat P.
aeruginosa [35].
I.1.3.1.2 Protection of antibiotic producers
In antibiotic producing bacteria, there lies a cellular mechanism that
protects the producers from being vulnerable to their own lethal chemicals. The
majority of natural products are made by enzymatic machinery which is
coordinated with trans-membrane protein pumps. These proteins pump out the
chemical before it accumulates in the cell at harmful concentrations. For
instance, oleandomycin is produced in its glycosylated form (inactive) which only
becomes activated once it is secreted in the environment. Furthermore,
erythromycin producers modify their own target by demethylation of adenine in
23S rRNA to prevent it from binding to 50S ribosomes with high enough affinity
[36].
I.1.3.2 Synthetic production
As mentioned in I.1.2.3.1, there are three major classes of synthetic
drugs: sulfa drugs, the quinolones, and the oxazolidinones. Synthetic compound
discovery is of keen interest based on the assumption that there will not be any
pre-existing resistance gene associated to them in nature. The modern approach
to finding synthetic antibiotics is a combination of synthetic biology and
metabolic engineering [37]. Design, modeling, synthesis and validation are four
key aspects of the antibiotic production process. Here are the details of genomic
based approach that combines high throughput technology, DNA technology,
computational methods and synthetic chemical library building techniques.
10
I.1.3.2.1 Genomic based approach
Invention of faster and more efficient tools for genomic sequencing has
generated 150 new microbial genomes in 2010 [38]. To select a potential
antibacterial target, researchers look for conserved ORFs across the pathogens
that may still be worth attacking. Secondly, it is preferred that conserved ORFs
are absent from complex eukaryotes to reduce the toxicity to humans. These
two criteria can be determined using bioinformatics, though a third criteria need
to be determined experimentally. This final criterion is that ORFs must be
essential in one or more pathogens which are usually determined via creating a
functional knockout. The genomics approach to antimicrobial drugs involves
target selection, screen development, compound lead identification and lead
optimization [39]. Thus far, the genomic approach has not been quite successful
in providing new leads due to a limited understanding of molecular targets,
challenges in optimization of enzyme inhibitors and the limitation of compound
screening libraries [40].
I.1.4 Antibiotic resistance
I.1.4.1 Resistance is here to stay
Antibiotic usage and emergence of resistance are two sides of the same
coin. Only two years after the introduction of penicillin for the treatment of S.
aureus infection in 1940, the emergence of resistant strains was reported [41].
This phenomenon made us realize that our anticipated triumph over
microorganisms was temporary. To win this ongoing battle against resistance,
and to combat the complex survival strategies employed by bacteria, there is
immense pressure on the industry to develop new drugs or modify existing
drugs. In the United States, hospital based infections caused by the following six
pathogens are considered highly severe due to their resistance to multiple
antibiotic drugs. They are collectively termed “ESKAPE” pathogens, which include
Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumanii, Klebsiella
11
pneumoniae, Pseudomonas aeruginosa and Enterobacter species [42]. There are
three main types of resistance: 1) intrinsic resistance 2) acquired resistance and
3) adaptive resistance. In intrinsic resistance, bacteria are naturally resistant to
antibiotics due to their biology, where as acquired resistance occurs after the
bacterium comes in contact with an antibiotic [43]. In contrast to the prior two
phenotypes, adaptive resistance is reversible. It is a non-mutational, transient
resistance which is induced in the presence of a specific trigger such as pH,
anaerobiosis, antibiotic, cation levels, biofilm formation or swarming motility
[44]. For the purpose of this thesis, I will focus only on intrinsic and acquired
resistance types.
I.1.4.2 Intrinsic Resistance
Despite of general understanding that resistance exclusively emerges as a
result of clinical use of antibiotics, recent overwhelming evidences suggest that
antibiotic resistance is much more ancient in nature. Analysis of DNA sequences
obtained from Late Pleistocene permafrost sediments (30,000 years old),
showed highly similar sequences to vancomycin resistant genes (VanHAX) [45].
Researchers also reconstructed protein from ancient genetic sequences and
proved that it encodes a functional enzyme with binding site D-alanyl-D-lactate
which is responsible for vancomycin resistance (see I.1.2.3.3i and I.1.4.4-5). In
addition, structure based phylogeny dates back the origin of β-lactamase
enzymes to two billion years [46]. Whether it is undisturbed cold-seep
sediments on the ocean floor, remote Alaskan soil or pristine freshwater
environment near Brazil; resistance conferring genes and/or MDR bacteria are
found ubiquitously in presumably “antibiotic-free” environments [47-49].
I.1.4.3 Molecular epidemiology of acquired resistance
Antibiotic resistance genes can be divided into ‘pre-antibiotic’ and
‘antibiotic’ periods. Macroevolution of resistance in nature over millions of years
is accelerated in hospital settings. Bacterial population in the microenvironment
12
of medical facilities is under intense selective pressure of antibiotics. Since, a
typical frequency of error in DNA replication is 1 in 107 bases and a bacterial cell
can divide in as short as 20-30 min, there is a likelihood of 10 or more mutants in
a population of 108 bacteria. In an E. coli genome of 3000 genes, roughly 0.3% of
genes will have a spontaneous point mutation [17]. If one of these genes
happens to be the target for an antibiotic, the existing mutation gives the
bacteria selective survival advantage that enables it to grow and dominate the
culture while its sensitive neighbors die. In addition to selection, the transfer of
genetic material is another powerful mode of acquiring resistance.
There are two modes of resistance transmission: vertical and horizontal.
When a mutation is acquired in a chromosome (as explained above), it is known
as vertical transmission. Horizontal transmission can be further categorized into
three mechanisms: conjugation (bacteria to bacteria), transformation
(environment to bacteria), or transduction (virus to bacteria) [50]. Material used
for horizontal transmission can be of different types. In some cases, the
‘advantageous’ genes with point mutations can be grouped on transposable DNA
elements known as “transposons.” Transposons are mobile genetic elements
that can transpose between DNA sequences element on both plasmids and
chromosomes [51]. For example, genes responsible for VanA phenotypes in VRE
cells are on a transposon embedded in a plasmid. Integration of these plasmid
DNA elements into specific attachment sites on chromosomes creates antibiotic
resistant islands as found in MRSA [52]. This allows multiple resistance genes to
be maintained and spread collectively together through bacterial populations. As
a result, resistance to one antibiotic in one species of bacteria can rapidly make
another species resistant to the same antibiotic without it ever having to be
exposed to it. The development of antibiotic resistance is not a matter of if but
rather a matter of when [53], hence there needs to be a constant supply of new
classes of antibiotics in the market.
13
I.1.4.4 Cellular modes of acquiring resistance
Acquired resistance is of greater medial concern among health
professionals. When exposed to antibiotics, bacteria rapidly adapt and develop
mechanisms to protect themselves. There are five major mechanisms through
which bacteria acquire resistance [Figure 1.5]:
1) Denial of Entry: Majority of antibiotics enter the cell via specific water
filled channels embedded in the outer membrane known as porins. Down-
regulation of porin generating genes, mutations in the binding sites of porins or
an absence of specific porins in a specific cell can make the bacteria resistant to
the antibiotic. For example, the absence of D2 porins in Pseudomonas
aeruginosa makes them resistant to imipenem [43]. Furthermore, bacteria can
alter their cell wall structure as such and down regulate receptors so that the
binding site of the antimicrobial agents is no longer a viable option [54].
2) Drug Efflux: In order for antibiotics to be potent, they must accumulate at
certain concentrations in the cell. As observed in the case of tetracycline
resistance (eg. tetA), bacteria increase the production of efflux pumps that
actively expel a wide range of substrates [55]. Both gram-positive and gram-
negative bacteria intrinsically process similar transporters to pump out lipophilic
and amphipathic molecules as well as intrinsically made natural antibiotics. This
mechanism does not prevent the entry of the drug but it decreases intracellular
concentration of the drug by increasing its efflux.
3) Antibiotic modification: In this mechanism, bacteria inactivate the
antibiotic by modiinng the molecule. Antibiotics with a four membered β-lactam
ring (ie. penicillin and cephalosporines) are hydrolyzed by over 200 types of β-
lactamases [43]. These ubiquitously found enzymes can be inhibited by clavulanic
acid [56] which is co-prescribed with β-lactams in the case of resistant strains.
Functional group transfer (e.g. chloramphenicol) [57], functional group
modification (e.g. aminoglycosides) [58], redox reactions (eg. tetracyclines) [59]
14
and proteolysis (e.g. antimicrobial peptides) [60] are some of the examples of
mechanisms by which antibiotics are modified in the bacterial cells.
4) Metabolic bypass: Metabolic bypass is the ability of bacteria to overcome
the effect of antibiotics by either over production of the chromosomal copy of
the enzyme or by synthesizing a replacement copy of the enzyme provided by
resistance plasmids [54]. This replacement enzyme is insensitive to the
antibiotic, yet performs the same reaction as the chromosomal copy of the
enzyme. Resistance to sulfonamides and trimethoprim is commonly associated
with this mechanism [61, 62]. Alternative penicillin binding protein (PBP2a)
produced by MRSA, and alternative cell-wall precursors D-alanyl-D-lactate in VRE
are also examples of metabolic bypass [63, 64].
5) Target site modification: The majority of antibiotics bind to specific
residues of a peptide chain or nucleic acid sequence. Minor changes in the amino
acids or DNA/RNA sequence can drastically affect the potency of an antibiotic.
For example, loss of one hydrogen bond from D-alanyl-D-alanine to D-alanyl-D-
lactate results in 1000 fold decrease in affinity between vancomycin and
peptidoglycan [65]. Resistance emerging due to this mechanism rapidly spreads
via horizontal gene transfer. Therefore, cross-resistance to the macrolide,
lincosamide, streptogramin, ketolide, and oxazolidinone (MLSKO) group of
drugs among various gram-positive and gram-negative species is of a great
concern [66].
I.1.4.5 Combating antibiotic resistance
The spread of antibiotic resistance is forecasted to increase in the near
future due to rapidly changing climate conditions and increasing global migration
[67]. There need to be many short-term and long term measures in order to
limit the emergence and spread of resistance. Conservative and rotating use of
antibiotics, investment in precise diagnostics and improved sanitation methods
are among the top priority actions required against resistance. To maintain long
term interest of industry in development of new antibiotics, there needs to be a
15
strong public and private partnership, extension of patent periods, and various
‘push and pull’ incentives [68].
I.1.5. Light at the end of the tunnel
Clinical resistance is a complex phenomenon because it is influenced by
many different variables, such as type of bacterial species, the organ or tissue
being infected, overall distribution of antibiotics in the body and its local
concentration at the site of infection, as well as immune state of the patient
[43]. Where one group is highly pessimistic about the future of antibiotic use
due to resistance [69], there exist a group of optimists about the development of
new antibiotics [70]. There are new targets being explored such as bacterial cell
division, type III secretion systems, bacterial signaling and host response
pathways. There has been a huge leap in applying new strategies for discovery
such as marine and plant-based compound libraries, resurrection of
undeveloped drugs, combinatorial drugs and passive immunization. There are at
least 16 new drug molecules in clinical trials with novel mechanisms of action
[71]. In addition, due to increased awareness there are ongoing improvements in
funding, incentives and legislation. Recently, the US congress has proposed a 2-
year wildcard patent extension for drugs that are used for military or
antiterrorism purposes [72]. The lesson we must learn from the history is that
failure to use antibiotics in a frugal manner will render any number of new drugs
useless in a short while. Consequently, like any other natural resource,
antibiotics should be valued as a finite source.
16
I.2 Tirapazamine
I.2.1 Origin of the pro-drug
Accordingly to the 2008 WHO report, the cases of cancer are expected to
double by 2020 and tripled by 2030 [73]. With 12.7 million new cancer cases
reported in 2008, the global health burden due to cancer is equal, if not more, to
that of microbial infections [74]. Therefore it is no surprise that majority of new
and interesting drugs with novel mechanisms of action are first applied for
combating this modern day disease globally responsible for 21,000 deaths per
day [74]. One of those drugs is Tirapazamine (3-amino-1,2,4,-benzotriazine-1, 4-
dioxide, TPZ, SR-4233, SR 259075, WIN 59075) which is extensively researched
for its anticancer activity in various types of cancers as a hypoxic cell sensitizer.
Since the discovery of hypoxic microenvironment in tumors in mid 1950s
[75, 76], drugs that can selectively target hypoxic cells are of great interest.
Hypoxic environment is caused by reduced or obstructed blood flow and oxygen
supply. Due to high growth rate of tumor cells, it is not uncommon for tumors to
have incomplete or inconsistent vasculature. Blood vessels are the medium by
which cells receive anti-cancer drugs, therefore, decreased blood flow renders
chemotherapy ineffective [77, 78]. Hypoxic cells also show resistance to ionizing
radiation, because radiation requires oxygen to form ROS which ‘seals’ the
damage initiated by radiation. Hypoxic cells are capable of repairing the
radiation initiated damage possibly through non-protein sulfhydryls [79, 80].
Consequently, cells with inefficient oxygen delivery are resistant to ionizing
radiation [76]. Furthermore, hypoxia induces hypoxia-inducible transcription
factor 1 (HIF-1) [81] and amplifies the amount of mutated p53 which further
increases the chances of tumour metastasis [82]. Thus, hypoxic cells in solid
tumour are intrinsically resistant and present a great challenge in cancer
treatment [83]. Therefore, development of hypoxia-selective pro-drugs or “redox
sensitizers” has been of great interest in cancer therapeutic research.
17
Traditional chemotherapeutic agents are poor discriminators of
neoplastic cells, retain narrow therapeutic index and cause severe damage to
normal cells. In order to overcome these limitations, the concept of pro-drug
was introduced. The term “pro-drug”, was first coined by Albert in 1950s,
described as “compounds that need to be transformed before exhibiting their
pharmacological action [84].” Increasing the bioavailability of antitumor agents
and delivering an antitumor agent specifically to target cells are two main
purposes of a pro-drug [85]. The clinical potential of TPZ was highlighted by
Zeman and colleagues in 1986 as a part of drug discovery program sponsored by
SRI international and Standford Univeristy [86]. However, initial synthesis of TPZ
was carried out by R. F. Robbins and K. Schofield as a derivative of 1,2,4-
Benzotriazines in 1957 [87]. The most important aspect that immediately put TPZ
in the spot light of cancer research was its relatively high hypoxic cytotoxicity
ratio (HCR). HCR is defined as “a ratio of drug concentrations under aerobic (air)
to hypoxic (N2) conditions to yield approximately the same survival [86].”
Depending on the cell lines used, TPZ showed a large differential cytotoxicity
ratio up to 300 for SCCVII mouse tumor cells [88] and between 15-150 in various
human cell lines [86, 88]. Alas, in vivo study in tumour bearing mice has shown
only three fold difference in cell killing under hypoxia [89]. There are several
factors responsible for the large variations in HCR of TPZ including, the
population of hypoxic cells in the tumor of subjects, the level of hypoxia
achieved in laboratory experiments, and the percent of active TPZ reaching the
tumors [90]. Owing to some extremely promising preliminary results, TPZ was
the first drug to be introduced into clinic for its activity against hypoxic cells [83].
This thesis is possibly the first record of utilization of TPZ in prokaryotic systems,
thus all the findings mentioned from here onwards are carried out in either in
vitro system or some form of eukaryotic system, unless motioned otherwise.
18
I.2.2 Chemical structure and properties
The chemical name of TPZ is 1,2,4-Benzotriazin-3-amine, 1,4-dioxide with
molecular formula C7H6N4O2. As part of benzotriazine (BTZ) family of
compounds, the core structure of TPZ consist of a benzene ring adjacent to a
1,2,4-triazine ring [Figure 1.6]. Two nitrogens at the position 1 and 4 are oxidized
to create an aromatic di-N-oxide. 3-amino functional group at 3rd carbon of
triazine ring specifically places TPZ into the BTZ class of molecules with 3-amino
group [Figure 1.6]. In addition to antitumour activities [91], BTZ derivatives have
been observed to possess antimalarial activity [92], antifungal activities in vitro
against phytopathogenic fungi [93], anticonvulsant properties [94], and ability to
inhibit Ableson tyrosine kinases [95]. The BTZ molecule itself is colourless,
however when both ring nitrogens are oxidized, as in the case of TPZ, it produces
a bright reddish-orange powder. TPZ possesses water solubility of 1430mg/L at
20°C [96]. TPZ solution is light sensitive and has photodegradation half-life of
1.95 days in water [96]. Therefore, it is important to protect both the powder
and the solution from light exposure while performing experiments. There have
been numerous structural modifications engineered on TPZ molecule over the
past two decades in order to improve its clinical potential. Table I.2 summarizes
a subset of important modifications and their effects on molecular properties of
TPZ.
I.2.3 Mechanism of preferential toxicity
Among many drugs tested for their selective toxicity to hypoxic cells, TPZ
emerges as a drug with unique combination of characteristics that fits perfectly
for targeting tumour hypoxia. TPZ is toxic in the same range of oxygen partial
pressure (0-36 torr) as found in radiation resistant tumour cells (<30 torr) [97]
unlike nitroimidazoles and mitomycin C which are active at 3 torr [98] and 0.3
torr [99], respectively. Although HCR of TPZ varies depending on the cell line (as
mentioned in I.2.1), it is comparable to that of misonidazole [100] and aziridine
19
derivatives of nitromidazoles [101]. Comparatively, what gives TPZ an edge
among other drugs is its effectiveness at a much lower concentration (5μM) than
preceding molecules. The unusual activity curve of TPZ is never saturated and
inversely proportional to the oxygen availability. There is a 2000 fold difference
in activity between hypoxia and 1mM oxygen [102]. The mechanism of this
preferential toxicity, though thoroughly studied, is only partly understood. TPZ is
a pro-drug that requires activation by cellular enzymes to carry out medically
relevant processes. Once activated by one-electron reduction, the resulting
radical species is thought to damage DNA in direct or indirect manner. The
recent understanding concerning each step of the mechanism is summarized in
the subsections below.
I.2.3.1 Pro-drug activation by reducatases
Among several cellular enzymes capable of reducing TPZ [103, 104],
NADPH: cytochrome p450 reductase (EC 1.6.2.4) plays a central role in
microsomal reduction [105-107]. In addition, enzymes such as cytochrome
p450, NADH, xanthine oxidase [108, 109], aldehyde oxidase and DT dia-phorase
[110] are known to reduce TPZ [111]. Since DNA is found mainly in the nucleus,
cellular sensitivity of TPZ is highly dependent on the activity of nuclear
reductases [112, 113]. Given that this thesis focuses on utilization of TPZ in
bacterial systems, the difference in activation linked to sub cellular localization is
of little importance. In yeast, overexpression of NCP1 (human p450 orthologue)
has been shown to increase sensitivity of yeast strains to TPZ [114]. Therefore, if
TPZ was to be used as an antibiotic, we can safely predict TPZ to have similar
reductive mechanism of activation in bacteria.
I.2.3.2 Oxidative damage caused by TPZ
There are three main theories for the exact species that carries out the
oxidative damage.
20
i) TPZ radical anion (TPZ●) itself is capable of causing single and DSB in
plasmid DNA [109]. Evidences supporting this theory were gathered via pulse
radiolysis and xanthine oxidase. Radiation chemical reduction experiment
showed lifetime of TPZ● up to many milliseconds under hypoxia, suggesting
enough time for it to accumulate at μM concentration and diffuse into cells.
ii) A more recent model suggests dehydration of TPZ● to produce BTZ
radical (BTZ●) [115]. BTZ● is not only capable of DNA damage, but it can also
oxidize the TPZ molecule itself [115-118]. Though, BTZ● on its own cannot cause
sufficient damage to DNA because one electron reduction potential (E1R) of BTZ●
(1.31V) is much less than what is required (1.6 V) for direct oxidation of guanine
in pyrimidine bases [119, 120].
iii) Widely accepted and extensively studied by Dr. Kent S. Gates from
University of Missouri is the theory that TPZ● decays to hydroxyl radical (●OH)
[121-125]. The products resulting from TPZ-mediated damage to purine,
pyrimidine and deoxyribose components of DNA are similar to the products
generated by interaction of ●OH to DNA. Furthermore, studies with electron
paramagnetic resonance and density functional theory also support release of
●OH from TPZ [126-128].
I.2.3.3 Futile cycle of enzymatic reduction under aerobic condition
TPZ● exist in equilibrium with its neutral form. The equilibrium shift is
governed by oxygen availability. The dramatically low cytotoxicity of TPZ, under
aerobic condition, is owing to this back oxidation of the radical species to non-
toxic parent molecule in the presence of oxygen [Figure 1.7]. This otherwise
futile cycling, results in a concomitant production of highly toxic superoxide
radical (O2•) [126, 129-131]. Though, O2• does not prove to be toxic to the cell
because of the armamentarium of enzymes such as superoxide dismutase,
catalase, glutathione peroxidase, and peroxiredoxins provide cell with a robust
defense system against superoxides and ROS [132-134]. Hence, hypoxia
21
generated TPZ● proves to be much more cytotoxic than the superoxide radical
generated under aerobic condition.
I.2.3.4 Secondary metabolites
After causing radical mediated damage to macromolecules, one electron
reduction product of TPZ gains another electron in an irreversible step to form a
detectable, non-toxic, mono-N-oxide, SR 4317 in vitro [135], in mice [136], and in
humans [137]. In fact, the lack of toxicity of SR 4317 in either aerobic or
anaerobic cells is one of the main evidences to support that the intermediately
formed TPZ● is toxic. Nevertheless, SR 4317 plays a secondary role in fixating
initial DNA damage caused by either ●OH or BTZ● and facilitates cytotoxicity of
TPZ specifically in hypoxia [138]. SR 4317 can be formed by radical
disporportionation, two-electron reduction of TPZ by the enzyme DT-diaphorase
[110] or by hydrogen abstraction from cellular macromolecules. Another non-
toxic, four electron reduction product SR 4330 has also been reported to form in
a relatively smaller amount [135, 139-141].
I.2.3.5 Macromolecules as a target for TPZ
It is widely accepted that the active radical species derived from TPZ
removes a proton from DNA forming DNA radicals [140, 142]. In addition, TPZ
has been known to play a role in fixing these DNA radicals by acting as a
substitute for O2 [143-145]. As a result of this damage fixating action of TPZ,
single and DSB are created in DNA. Upon sufficient DNA damage, a p53 based
apoptosis mechanism is activated leading to cell lethality [146]. The DNA damage
caused by TPZ requires homologous recombination (HR) pathways to repair it
[147]. The general mechanism of HR is well conserved among all kingdoms. In
prokaryotes, HR involves strand-invasion and strand-exchange catalyzed by RecA
[148]. Although RecA plays a central role, additional two dozen or more proteins
are involved in this process including but not limited to RecBCD, RecE, RecF,
RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins
22
[149, 150]. Cells that are deficient in HR function can be expected to be
hypersensitive to drugs causing DNA damage.
I.2.3.5.1 Topoisomerase II
According to recent understanding, DNA DSB are protein associated [151,
152]. DNA cleavage is thought to result from stabilization of topoisomerase II
(topo II) complex by the active radical species [153]. Topo II, known as DNA
gyrase in bacteria, has two subunits (A and B) each containing an ATPase
domain. A conserved tyrosine residue in the active site forms covalent
phosphotyrosyl bond with the phosphate backbone of DNA. This creates a DSB
(G segment) through which another strand is able to pass through (T segment)
[154]. Religation of G segment completes the enzymatic activity of DNA gyrase.
This is a type II topoisomerase that unwinds positively supercoiled DNA by
changing their linking number by 2 [Figure 1.8]. The introduction of negative
supercoils in DNA is vital for progression of replication fork, and transcription.
TPZ has a similar topo II inhibition mechanism as the drug Adriamycin and
etoposide [155], except the TPZ cleavable topo II complex is less stable than the
complex found with etoposide [153]. TPZ is also known to inhibit cellular
accumulation of HIF-1α at the translation stage, independent of topoismerase IIα
inhibition in human cancer cells [156]. DNA gyrase has been a popular target
among antibiotics, specifically fluoroquinolones. Details on quinolone mediated
mechanism of inhibition have been explained previously (section I.1.2.3.3.3).
I.2.4 TPZ as a potential anticancer drug
Cancerous cells are a combination of both well oxygenated as well as
hypoxic cells. TPZ is not sufficiently toxic under aerobic condition, particularly at
the doses that are tolerable to humans. Therefore, TPZ was never expected to
have antitumor efficacy on its own. TPZ has been tested in association with
ionizing radiation and various chemotherapeutic drugs such as platinum
compounds (cisplatin and carboplatin), cyclophosphamide, melphalan, and
23
taxanes [157]. The efficacy of TPZ is highly dose and schedule dependent, and
performs best when it precedes the other cancer treatments [158, 159].
In order to overcome tumor hypoxia a range of compounds from N-
dioxies, quinines, nitro-aromatics, and metal complexes have been studied [160],
however, TPZ is the most advanced hypoxia activated agent evaluated in clinical
trials. In a phase III trial, addition of TPZ with cisplatin for advanced NSCLC
patient has shown improved survival compared to cisplatin alone [161, 162].
Recent phase I and phase II studies by South Oncology Group (SWOG) in small-
cell lung cancer patients with TPZ in combination with thoracic radiation,
cisplatin, and etoposide has shown increased median survival of patients [163,
164]. In addition, TPZ has shown promising results in phase II trials of advanced
HNC [165-167], metastatic melanoma [168], cervical cancer [169, 170], ovarian
cancer[171], glioblastoma [172] and several other solid tumors. However, when
it comes to phase III trials in NSCLC [173] and HNC [174], TPZ has failed to show
overall positive results. A significant factor contributing to the disappointing
clinical result is our limitations in classification of hypoxic tumor cells. There
needs to be more definite markers of hypoxia through which we can identify
patients with significant population of hypoxic tumor cells and only those
patients should be recruited for future clinical trials with TPZ.
Maximum tolerated doses (MTD) of TPZ have been 220-390 mg/m2 when
associated with chemotherapeutic agents and 160 mg/m2 (equivalent to 250mg),
12 times a week, when coupled with radiation [165, 175, 176]. The side effects
observed in patients under clinical trial for TPZ include nausea, vomiting,
diarrhea, weight loss, skin rash, muscle cramps, tinnitus, acute reversible hearing
loss [177], visual disturbances, cardiac ischemia and transient loss of
consciousness [178]. These effects are highly dose dependent and, as we
become aware of the dose range necessary for treatment, we expect to lower
the toxicity profile of TPZ. Confronting further details of each clinical trial would
be out of the realm of this thesis, however, the data have been elegantly
24
summarized and tabulated in many publications [178-180]. In summary, TPZ was
a highly anticipated drug as a radio sensitizer since it was expected to
complement radiation killing of aerobic tumor cells [181]. TPZ has shown
contradictory results in clinical trials, and has failed to replicate positive phase III
NSCLC clinical trial results to date. Consequently, the fate of TPZ as an anticancer
drug is heavily dependent on the outcome of future clinical trials underway in
NSCLC, HNC and cervical cancer.
I.2.5 Anticancer drug to serve as an antibiotic
One of the novel approaches to introduce new classes of antibiotics and
combat microbial resistance is to find new applications for old drugs and their
derivatives. There have been number of antibiotic drugs that are now being
considered as antitumor agents. For example, salinomycin [182, 183],
resistomycin [184] , non-antimicrobial tetracyclines [185], beta-lactam
derivatives [186], and macrolies [187]. There are plenty of molecules that are
referred to as antitumor antibiotics because they actively target both microbial
system as well as tumor growth by targeting DNA. Molecules such as olivomycin
A [188], mithramycin [189], and approximately 20 enediyne class of molecules
[190, 191] are known as antitumor antibiotics. Considering this and the fact that
BTZ class of molecules has previously shown antimalarial and antifungal
activities, it is highly probable for TPZ to have antibiotic properties. The objective
of this project was to investigate TPZ as a broad-range antibiotic and obtained its
basic mechanistic insight in bacteria.
25
I.3 Clostridium difficile Infection
I.3.1 Global epidemiology of Clostridium difficile Infection (CDI)
In the summer of 2011, Clostridium difficile (C. difficle) outbreaks in
southern Ontario hospitals had claimed at least 31 lives [192] and 300 cases
were diagnosed in the month of June alone. With twelve new cases in Kingston
general hospital in August 2011, there is an increasing threat of the C. difficile
epidemic in Canada. In the United States, approximately 15,000 to 20,000
patients die from C. difficle infection (CDI) annually, and 333,000 initial cases
have been reported due to CDI, which is costing the health care system up to
$3.2 billion dollars per year [2]. Although the number of CDI incidences has
started to decrease in England and the Netherland, in the U.K. alone, 3,933
death certificates have mentioned CDI in 2009 [193, 194]. In a Europe based
survey, mortality rate due to CDI is estimated to be 40% [193]. There have been
reports of CDI from Japan, Hong Kong, Korea and Australia, though data
availability from Asia, Africa and the Middle East has been inadequate.
I.3.2 C. difficile infections are difficult to treat
C. difficile is one of the most common hospital infections associated with
iatrogenic complications in the developed world. It is a gram-positive, spore
forming, rod shaped bacteria [Figure 1.9]. C. difficile is ubiquitously found in the
normal intestinal microbiota of 1-3% adults and up to 80% of healthy infants
[195]. Albeit, this rate increases to 10-30% in hospitalized patients [196]. Not all
the strains of C. difficile are toxic, although strains producing toxins can still
prevail as an asymptomatic colonization.
C. difficile gets its name from the Greek word ‘kloster’ (spindle) and the
Latin word ‘difficile’ (difficult). This bacterium is was given this name because it
was difficult to isolate and grow in culture [197]. The growth of C. difficile in
liquid or solid media has a signature ‘horse-pee’ like smell. The vegetative
26
growth of this organism is restricted to anaerobic environments, though spores
can survive hot, acidic and other harsh conditions. Spores of this bacterium are
resistant to standard ethanol based sanitizers which impose greater risk of
spreading the infection. Chlorine and hydrogen peroxide containing disinfectants
have been suggested for CDI [198, 199].
I.3.3 C. difficile pathogenicity
First described in 1935, C. difficile was not considered pathogenic until its
association with pseudomembranous colitis (PMC) in 1978 [200]. This is about
the same time as the introduction of broad spectrum of antibiotics in clinical
practice. Improper stewardship of antibiotics is claimed to be the primary factor
in triggering CDI. Specifically, the use of broad spectrum antibiotics, such as
cephalosporins, clindamycin and fluoroquinolones, have been associated with
increased rates of CDI in the 21st century [201]. Broad spectrum antibiotics
disturb the intestinal microbiota equilibrium, killing most of the other bacteria
and allowing growth of resistant C. difficile strains.
The drastic fourfold increase in nosocomial CDI cases and their severity
from 1998-2004 was first reported in Quebec, Canada [202, 203]. This was in
part due to the emergence of hypervirulent, fluoroquinolone resistant, strain
Nap1/027/BI/III (North America PFGE pulsotype 1 = NAP1. PCR ribotype=027,
restriction endonuclease analysis group BI, and toxinotype III). There are many
different ways to characterize the strain but PCR ribotyping is the most common
method. This method takes advantage of polymorphism in the 16S-23S
intergenic spacer region to type C. difficile. Nap1 epidemic has generated an
immense amount of interest in C. difficile research, and is believed to be the
major cause of the increasing severity and frequency of CDI both in North
America and in Europe. In a recent survey of 389 patient isolates in Europe, 65
different PCR ribotypes were identified. Out of these only 5% PCR-ribotypes
were 027, other common types are 014/020 (16%), 001 (9%), 078 (8%) [193].
27
Newer strains are more virulent and raise a great deal of concerns on combating
C. difficile in the future.
I.3.4 CDI pathology
C. difficile is the most common cause of infectious diarrhea in hospital
settings. Significant diarrhea is usually the first symptom for CDI, clinically
defined as three or more stools per day for one to two days [204, 205]. The
symptoms can range from mild to severe diarrhea, fulminant PMC, toxic
megacolon, intestinal perforation, sepsis, multi organ failure, and ultimately
death [206]. The complication will depend on how early the infection has been
identified, how virulent of the strain is and patient’s initial response to the
antibiotic therapy. Moreover, there have been 22 newly reported cases of C.
difficile enteritis [207]. According to this report, C. difficile not only affects the
colon, but it can also affect small intestine. Since there is a lack of awareness
regarding the enteritis aspect of C. difficile, it has a high mortality rate (40%) due
to lack of timely treatment [207]. Furthermore, patients with inflammatory
bowel disease (IBD) are at a higher risk of developing CDI since they are more
prone to C. difficile colonization [208]. Additionally, there are extra-colonic
manifestations of CDI which include enteritis, arthritis, visceral abscess
formation and pericarditis [209].
There is a unanimous consensus on improper antibiotic use being the
most important risk factor in CDI. Therefore, it is safe to suspect CDI in anyone
that develops diarrhea for up to 8 weeks after they have completed the course
of antibiotics [210]. Other risk factors include, age (65 or older), duration of stay
in hospital, exposure to other patients with CDI and any other conditions that
may affect colonic flora [201]. Specifically, the use of the following antibiotics is
considered high risk for CDI: cephalosporins, clindamycin, moxifloxacin,
gatifloxacin and levofloxacin [211]. There has been some debate in the literature
whether to consider proton pump inhibitors (PPI) and H2 blockers as risk factors
28
[212]. Although, vegetative cells of C. difficile can survive longer in a low acidic
environment, the spores - the most observed mode of transmittance, are highly
resistant to gastric acid to begin with. While CDI is mainly a nosocomial
pathogen, recent reports from the USA, Canada and Europe suggest a possible
increase in community acquired CDI [213]. The classic risk factors described
above are not sufficient in identifying community acquired CDI since half of the
patients over 65 admitted to hospital had no previous antibiotic exposure [214].
Therefore, further research is required to identify risk factors for community
acquired CDI.
I.3.5 Mechanism of action
Only C. difficile strains producing toxins are pathogenic. Although there
are many virulence factors that contribute to pathogenicity, there are two major
secreted exotoxins that are highly potent: toxin A (TcdA) and toxin B (TcdB).
These high molecular weight proteins (308 kDa and 269.6 kDa, respectively)
belong to the large clostridial toxins (LCTs) group and are involved in mucosal
damage and inflammation. They act on target cells by modifying small GTPases.
The multi-modular structure of toxin A and toxin B is classified as the ABCD
model. A) biologically active N-terminal domain B) C-terminal binding domain C)
cysteine protease domain, and D) hydrophobic domain [194]. The polypeptide
repeats in the C-terminal binding domain are involved in receptor binding to
specific cell-surface carbohydrate receptors [215]. Receptor mediated
endocytosis of the toxin allows it to enter the cell. A low pH environment in the
endosomal compartment triggers conformational change in the toxin molecule
that allows the highly hydrophobic ‘D’ domain to form a pore in the
compartment so that the ‘A’ domain can be translocated in the cytosol. Only
enzymatic ‘A’ domain is released in the cell through autoproteolysis mechanism
which is capable of targeting Rho GTPases (Rho, Ras and Cdc42). Rho GTPases
play a crucial role in many signaling pathways, specifically, in regulation of the
29
actin cytoskeleton [216]. Disruption of the cytoskeleton leads to cell rounding
and cell death.
There is a debate in the literature on whether one or both toxins are
sufficient in causing virulence [217, 218]. Whereas, purified toxin A can cause CDI
symptoms in animal models, toxin B requires toxin A or previously damaged
intestine to cause similar symptoms [217]. These results are under question
because naturally occurring A-B+ virulent strains have been reported in clinical
settings [218, 219]. Regardless of these conflicting observations, there are strong
evidences to advocate that toxin B is about 1000 times more potent than toxin A
[220].
Recently, there has been research on ADP-ribosylating binary toxin (CDT)
which is produced by the hypervirulent Nap1 strain. CDT has been identified in
up to 17% of the strains. Its high potency is a result of increase adherence to the
epithelial cells by inducing microtubule protrusion at the cell surface [221]. The
binary toxin is similar to the iota toxin of C. perfringens and has a partial deletion
in the tcdC gene that makes TcdC protein dysfunctional. This suppresses down
regulation of toxin production and the strain is able to produce excess of toxins A
and B in vitro [206]. However, the claim of high level toxin production has been
challenged by evidence of Nap1 producing more spores and the polymorphism in
the binding domain of toxin B [222, 223].
I.3.6 CDI diagnosis methods
We do not have technology to detect C. difficile bacterium itself
specifically, though there is a secondary diagnosis of toxins present in the stool
of affected patients. These toxins can be detected by actinomorphic changes
caused by them in fibroblast cell lines, and these changes can be reversed by C.
sordellii or C. difficile antitoxin. This cytotoxin assay was considered the “gold
standard” since 1978, until introduction of Enzyme Immunoassay (EIA). EIA was
widely used because the reagents were commercially available, and results can
30
be quickly obtained without requiring much technical expertise. Unfortunately, a
study of 45 reports from 1991-2008 shows that this test is only sensitive in 75-
80% of cases [224]. Thus, it is likely to show many false negative results. To
improve sensitivity, presence of the metabolic enzyme, glutamine
dehydrogenase (GDH), can be tested, since GDH is almost exclusively present in
C. difficile [225]. However, this only detects colonization by C. difficile and
therefore can give false positive results. Recently, PCR based detection of tcdB or
tcdA genes from stool are becoming increasingly popular due to its high
sensitivity. Though, it can only detect 50-60% of the strains that produce toxins,
the presence of toxin can be determined with high accuracy. The test is
commercially available since 2009 and it is expected to supplant the EIA in most
laboratories [226]. Current guidelines recommend to first use EIA or GDH as a
screening test and then to confirm positive results by cytotoxin or PCR assay for
toxin detection [227]. In a comparative study of commercially available
diagnostic tests to toxigenic C. difficile cultures in seven different laboratories in
North America, real-time PCR (RT-PCR) was proved superior to all other tests
with 93.5% sensitivity, and 94.0% specificity [228].
I.3.7 CDI therapeutics
Antibiotic course of 14 days is the first choice of treatment for CDI. While
oral metronidazole is prescribed for mild to moderate CDI, for severe CDI
(Leukocytosis with a WBC count ≥15 × 109/L and/or a serum creatinine level ≥1.5
times baseline level), oral vancomycin is recommended. Vancomycin, being the
only FDA approved drug for C. difficile, has a low MIC of 0.75-2.00 ug/ml,
sufficient to inhibit 90% growth of the strains [229]. What puts vancomycin
ahead of other antibiotics is its high intestinal absorption rate. In stools, it can
reach the concentration of up to 3100 ug/g, and only 4-16ug/ml is required to
inhibit resistant strains in vitro [230]. There has been a post-hoc subgroup study
of 172 people with severe CDI which proves vancomycin to be “superior” to
31
metronidazole [231], though the study itself is not convincing [232, 233]. First of
all, they used 50% lower overall dose of metronidazole in the study compared to
recommended dosage by Infectious Disease Society of America (IDSA).
Furthermore, the method to evaluate ‘treatment failure’ was not adequate and
the specific reason for assignment of treatment failure was not reported. In the
same study, metronidazole and vancomycin both had similar rates of clinical
cure, overall recurrence, resolution and recurrence of diarrhea [231]. Therefore,
further evidence is required to safely conclude that the more expensive
vancomycin is superior to metonidazole. The minor advantage of using
vancomycin is that it achieves faster microbiological cure, which helps reduce
the detectable levels of C. difficile in the stool samples [234]. Overall,
metronidazole is a more affordable choice for mild to moderate CDI, though
IDSA guidelines recommend metronidazole only up to the first relapse of CDI,
because its prolonged use can cause neurotoxicity. Beyond the first relapse of
CID, vancomycin is strongly recommended.
There are two major challenges in the treatment of CDI: refractory CDI
(severe CDI that fails to respond to initial medical therapy) and recurrent CDI
(recurs after initial successful treatment). Following the treatment of both
metronidazole and vancomycin, there are 2-37% cases of recurrent CDI and rate
increases to 64.7% in patients with a prior relapse [201].
For complicated CDI, such as in patient with ileus, toxic colon or
megacolon, when oral delivery becomes difficult, intravenous delivery of
metronidazole is required because intravenous vancomycin cannot reach lumen
of the colon. Though, in some cases 500mg of vancomycin is administered
rectally by an enema every 6h [235]. Other antibiotics such as bacitracin,
teicoplanin, fusidic acid, nitazoxanide, rifaximin, and tigecycline have shown
similar efficacy to vancomycin [206]. Fidaxomicin is a new, highly anticipated,
narrow spectrum, macrocyclic class of drug with high specificity for C. difficile
32
[236]. It is proven superior to vancomycin in phase III trials since it preserves
native microbiota of the gut.
For refractory CDI, immune therapy can be used adjunct to antibiotic
therapy where intravenous immunoglobulin (IVIg) is administered. Another
increasingly successful treatment is fecal microbiota transplant, where enema of
stool from healthy individual is transplanted in patient with CDI. Patient with
‘fulminant’ CDI (acute and severe), specially with leukocytosis, elevated lactate
levels, with hypotension requiring vasopressors, or those who had prior
treatment with IVIg or IBD, are often recommended a surgical procedure
involving removal of all or a part of the colon (colectomy) [237].
I.3.8 Concluding remarks
CDI is a global phenomenon effecting thousands of people and costing
billions of dollars to the health care system every year. This is especially the case
in Canada, where we may be on the verge of a C. difficile epidemic and new
antibiotics to fight this pathogen are in urgent need. Existing antibiotics are not
effective against refractory and recurrent CDI and have a failure rate of 20-40%.
As it is evident in the case of Enterococcus bacterial strain, use of vancomycin
pose a great risk of emerging resistance in already challenging pathogens such as
C.difficile. Therefore, development of a new class of antibiotics against CDI is of
great importance.
33
II Materials and Methods
II.1 Strains
Wild Type strain: E. coli K-12 BW25113 strain was used as a wild type strain in this study, which is derived from the original BD792 strain.
Name Genotype Pedigree Source Reference
BW792 rpoS396(Am) rph-1 W1485 CGSC6159 from B. Bachmann
[238]
BW25113c rrnB3 DElacZ4787 hsdR514DE(araBAD)567 DE(rhaBAD)568 rph-1
BD792 via BW25083 [239]
Pro+ with P1kc on BW24321 [239]
[240, 241]
c BW25113 is lacI+ [242]and not lacIq as previously reported [241]. Gyrase Mutant strains (MIC experiment):
Strain[243] Relevant genotype
KD1397 DM4100 tolC6::Tn10 gyr+ (wild type)
KD2862 KD66 tolC6::Tn10 gyrA (S83L)
KD2864 KD1975 tolC6::Tn10 gyrA (A84P)
KD2866 KD1977 tolC6::Tn10 gyrA (D87Y)
KD2876 KD1909 tolC6::Tn10 gyrA (S83W)
KD2878 KD1911 tolC6::Tn10 gyrA (A67S)
KD2880 KD1913 tolC6::Tn10 gyrA (D87N)
KD2882 KD1915 tolC6::Tn10 gyrA (G81C)
KD2884 KD1917 tolC6::Tn10 gyrA (Q106H)
KD2932 KD1500 tolC6::Tn10 gyrB (D426N)
*tolC deficiency lowers antimicrobial MIC
C. difficile strains: Nap1, Nap2, Nap3, Nap4, Nap5, Nap6, CIP107932 and
ATCC700057. These are clinical isolates provided by the Montreal General
Hospital. They were originally obtained from Center for Disease Control and
Prevention, Atlanta. ATCC stands for American Type Culture Collection and
ATCC700057 is common WT strain used as a control in C. difficile assays. ‘Nap’
stands for North America PFGE pulsotype.
Staphylococcus aureus strains: Kindly provided by Dr. Brigitte Lefebvre from
Laboratoire de santé publique du Québec/ Institute national de santé publique
34
du Québec. Methicillin Sensitive Staphylococcus Aureus (MSSA): MA076688,
MA076723, MA076899, MA077045. MRSA: MA077046, MA077064, MA077074,
MA077085.
Reductase mutant strains: There were 197 single gene deletion strains of E. coli
K-12 (host strain: BW25113) tested from the ‘Keio collection’, Japan [242] [See
Appendix A]. This collection was created by insertion of a kanamycin cassette in
the place of ORFs of 3985 non-essential genes in E. coli K-12. The kanamycin
cassettes are flanked by FLP recognition target (FRT) sites which can be excised
by FLP recombinase on plasmid pCP20. In order to maximize the chances of
finding reductases involved in TPZ metabolism, keyword “reductase” search was
applied to the entire keio collection, Genobase.Version6 available on the
following website: http://ecoli.naist.jp/GB6/search.jsp. This resulted in the
identification of 191 genes, most of which were tested depending on the
availability (Appendix A). Additional genes were included as E. coli filtered hits of
basic blast search of human NADPH cytochrome p450 reductase protein
sequence (GI: 127139033; NP_000932.3) on NCBI http://blast.ncbi.nlm.nih.gov/.
The genes in the Protein-BLAST results that did not belong to 191 genes were, in
order of ranked hits: cysJ, moiC, yqcA, fldA, yrbG, yeeU (not included), ydbH,
mltb, hmp, and puuC. Additional details of all strains are listed in Appendix A and
can be accessed via the “JW” ID online at http://ecoli.naist.jp/GB6/search.jsp or
http://www.shigen.nig.ac.jp/ecoli/strain/nbrp/searchBrowseKeioCollection.jsp.
EckID represents E. coli K-12 strain gene accession number.
E. coli deletion strains: A subset of genes involved in homologoues
recombination tested via drug spotting assay: recA, recB, recC, recD, recF, recJ,
recN, recO, recQ and recR. These genes were also provided by the ‘Keio
collection’ and details are included in Appendix A2.
35
II. 2 Drugs and media:
Tirapazamine (TPZ) was obtained from Sanofi-Aventis US Inc (lot no. 2002C).
Stock solution of TPZ was prepared in water at no more than 10mM
concentration. It was stored at room temperature in dark for maximum of 28
days. For bacterial growth in media and on plate, standard Luria broth (LB) was
used. LB media contains 10% tryptone (EMD chemicals Inc), 5% yeast extract
(EMD chemical Inc), and 10% NaCl. As needed, bacto agar (BD Difco agar
granulated) was used as a solidifying agent for growth on solid medium. While
testing TPZ sensitivity, an appropriate amount of TPZ was added to the LB media
before pouring into 25mM deep polystyrene round plates.
II.3 Drug spotting assay
Specific bacterial strains were streaked on LB plate from -80°C freezer stock to
obtain single colonies. Next day, a single colony was picked with a sterile
toothpick to inoculate in LB medium. Cultures were incubated overnight at 37°C
and their optical density at 600nm (OD600) was measured the next morning.
Then, they were diluted to 0.001 OD in a total volume of 300ul double distilled
water (ddH2O). Another three 10X dilutions were made (OD of 0.0001, 0.00001
and 0.000001) using 0.001 OD as the highest concentration in a total volume of
250μl. 96 well plates were used for this procedure. Using a multichannel
pipette, 10μl (or 5μl for certain experiments) of each dilution was spotted on LB
plate with or without antibiotic. The plates were incubated at 37°C under
aerobic or anaerobic conditions as indicated.
II.3.1 Anaerobic Incubation
For anaerobic condition BBL GasPakTM 100 system was used (Cat No. 260627, BD
diagnostics, USA). It is a specially designed jar that can hold upto 12 25mm
plates. The anaerobic environment in the jar is created by adding a palladium
catalyst pouch (BD BBL GasPak plus, cat no. 271040) or the AnaroGen
atmosphere generation system (Oxoid, 2.5L AN0025A). In the BBL system, the
36
sodium borohydride tablet reacts with water and generates hydrogen. This
hydrogen reacts with oxygen in the jar, in the presence of palladium catalyst to
form water again (NaBH4 + 2 H2O = NaBO2 + 4 H2↑; 2 H2 + O2 + [Catalyst] = 2 H2O
+ [Catalyst]). In this system 4-10% carbondioxide is generated by a separate
mechanism (Sodium bicarbonate plus citric acid tablet) within 2h incubation at
35°C, to stimulate the growth of organisms that require CO2- (C3H5O(COOH)3 + 2
NaHCO3 + [CoCl2] = C3H5O(COONa)3 + 3 CO2 + 3 H2 + [CoCl2]). Compare to 6h it
takes in BBL GasPak system for the indicator to turn colourless, the new Oxoid
system reduces the oxygen level below 1% within 30min. The carbon dioxide
level will be 9-13% in the new Oxiod system. The Oxiod system claims to be
hydrogen free, catalyst free, and does not require addition of water. The detail
mechanism of Oxiod system has been kept confidential by the company, but it
utilizes ascorbic acid, which reacts exothermically with oxygen in the air. A few
strips that turn colourless in reducing environment are also added (BD BBL
271051, active ingredient: methylene blue) as anaerobic indicators. High vaccum
grease (Dow corning, 2021846-0807) is applied at the circumference of the jar
before closing the lid. Parafilm is applied outside on the seal for added security
towards air leakage. Plates were dried properly before being placed in the
chamber upside down. This arrangement prevents condensation from the
gaspak entering the plates.
II.4 MIC determination of gyrase mutants
MIC is determined as the lowest concentration of drug that will inhibit visible
growth of bacteria after overnight incubation. This can also be referred as
minimum bactericidal concentrations (MBCs). MICs are considered to be a ‘gold
standard’ for susceptibility of microorganism to a given antibiotic [244]. This
experiment has been done previously by the lab that has provided us with the
gyrase mutant strains resistant to fluoroquinolone. Therefore, we decided to use
the same method of evaluation. Fluoroquinolone resistant strains [243] were
grown overnight in LB media. The following day, the overnight culture was re-
37
inoculated at OD600 0.002 and the culture was grown up until log phase OD600 of
0.1. The log phase culture was added at initial dilution of OD600 0.002, in 2ml LB
with appropriate concentration of TPZ or Norfloxacin (Sigma N9890-1G, store 2-
8°C, FW 319.3). Norfloxacin served as a positive control along with WT strains as
an internal control for individual experiments. The range of concentrations
tested were 0.005ug/ml to 1.5ug/ml for Norfloxacin and 5μM to 20μM for TPZ.
Since, we will be using only ratio between WT and mutant strains to graph the
results, difference in individual units of the drug concentration does not affect
the observations [See Appendix B]. No drug control and sterility control
(medium only) were also included. Cultures were incubated for 16-24h and the
results were collected by visual inspection of growth.
II.5 C. difficile experiment
This experiment closely follows Clinical and Laboratory Standards Institute
guidelines. These guidelines are followed worldwide and are bound to produce
globally comparable results. Reference agar dilution procedure (Wadsworth
method) was used to test the sensitivity of eight different C. difficile strains to
TPZ (section II.1). Two fold serial dilution series of 128μg/ml to 0.0586ng/ml
concentrations were tested. The strains were inoculated for overnight growth in
freshly made Schaedler Anaerobe broth (Oxoid, pH 7.6±0.2) from two times
streaked plates (Pre-made plates of Colombia blood agar, 5% sheep blood,
Oxoid). To increase the chances of anaerobic growth, inoculation was performed
quickly after autoclaving the media because autoclaved liquids have extremely
low level of dissolved oxygen content due to exposure to high temperatures.
Using, Wilkins Chalgren Anaerobe broth (Oxoid, Cat No. CM0643), 0.5
MacFarland(MF) turbidity starndard suspension of overnight cultures was
prepared. Steers replicator (or multiple inoculator, Cathra Automated Inc., St
Paul, MN 55112 USA; Mod NO. 73330-3mM; Ser No. 94ER25) was used to
inoculate the culture on solid medium. The approximate volume delivered on
the plate by the 3mM tip of the steers replicator is 2.1-2.24μl [245]. The eight C.
38
difficile strains were used in replicates on the same plate. 100ul of 105 dilution of
the 0.5MF (McFarland standard) was plated on brucella blood agar plates for
CFU counts. Appropriate dilutions for CFUs were made in schaedler broth.
Further details on this protocol can be obtained from Methods for Antimicrobial
Susceptibility testing for Anaerobic Bacteria; Approved Standard-Volume 27
[246]. The experiment was performed in duplicates with No drug anaerobic
growth control, aerobic growth control, and 4-10μg/ml amplicilin antibiotic
control.
II.6 Time-Kill assays (Killing Curves)
Log phase E.coli wild type culture was subjected to 50μM TPZ (4X MIC). Aliquots
were taken at time 0, 2, 6, 24h. The dilution was made in 0.9% saline solution
and culture was plated on LB agar plates (not TSAII blood agar plates as
suggested by the original protocol). Incubation was done at 37°C. A spreading
rod and turntable were used to spread the colonies on the plates. Three
different dilutions were plated for each time point and the average CFU of
culture with and without TPZ has been plotted on the y-axis.
To obtain the growth curve under anaerobic condition, cultures were
grown in three different jars. This gives each jar enough time to establish
anaerobic environment. Oxoid AneroGen pouches were used, as they create an
anaerobic environment within 30min. The wild type strain was subjected to
10μM TPZ under anaerobic conditions, in order to see the killing effect within a
short time of 2h. 0, 2, 4, 6, 8, 24h time points were one by one taken from each
of the three jars and LB plates were incubated in aerobic conditions at 37°C to
observe the surviving colonies. For detail information on protocols in this section
(II.6), please refer to section 13A.3.11 of Current Protocols in Pharmacology
[247].
39
III. RESULTS
III.1 TPZ has antibacterial properties
In eukaryotes, TPZ has shown to be activated through cellular reductases
to a radical species that ultimately damages DNA. Therefore, if bacteria are
sensitive to TPZ, this drug can create a foundation for a line of new class of
antibiotics. Drug spotting assay gives a quick overview of the effect of the drug
on the growth of microorganisms. Wild type E. coli strain BW25113 was spotted
on solid agar plate containing various concentration of TPZ. The growth of E. coli
was observed after overnight incubation at 37°C in normoxic and hypoxic
condition. In normoxic condition, colony forming defect can be seen starting
from 6.5μM to 10μM TPZ [Figure 3.1a]. Remarkably, it requires approximately
25 times lower concentration (0.375μM TPZ) to fully hinder the growth of
bacteria under hypoxic condition [Figure 3.1b]. This antibacterial property of TPZ
inspired us to further investigate its potency in antibiotic resistant strains.
III.2 Multidrug resistant strains are sensitive to TPZ
Multidrug resistance is a growing threat to public health. There is a
pressing need for new classes of antibiotics with novel mechanism of actions.
TPZ has already been tested as anticancer drug; therefore its toxicity profile for
human body has already been established. In order to be approved as a novel
antibacterial agent, TPZ must prove to be superior to existing drugs in the
market. Hence, we decided to test sensitivity of fluoroquinolone resistance
strains, MRSA strains, and C. difficile strains that are currently difficult to treat.
III.2.1 Fluoroquinolone resistance strains are sensitive to TPZ
In order to test the sensitivity of fluoroquinolone resistant E. coli strains
to TPZ, MIC determination experiment was carried out. MIC is the concentration
of drug at which negligible or no growth is observed by visual inspection for
strains growing overnight. Strains contained single amino acid (AA) mutations in
40
QRDR region of gyrA and gyrB gene that makes them resistant to
fluoroquinolones. Norfloxacin was arbitrarily chosen to represent
fluoroquinolone class of antibiotics and utilized as a positive control for this
experiment. The results are represented as a bar graph with ratio of mutant MIC
to WT MIC on the y-axis and AA mutation on the x-axis [Figure 3.2]. The ratio of
MIC mutant to MIC WT suggests the sensitivity of mutant to the respective drug.
If the ratio is greater than 1 the mutant is resistant to the drug since it is able to
grow at a higher concentration of the drug compare to its WT equivalent. The
experimental results show that the gyrase mutant stains give high values of MIC
ratio for norfloxacin. S83L and S83W are highly resistant to norfloxacin while,
A67S is the least resistant strain to norfloxacin. When these same set of strains
are tested with TPZ, MIC ratio is observed to be close to 1. D87Y, A67S, D87N
and Q106H shows MIC ratio of little slightly higher than 1, though it is nowhere
comparable to norfloxacin values. The G81C strain is the most sensitive to TPZ
and shows MIC ratio of much less than 1. Thus, it is safe to conclude that gyrase
mutant strains that are resistant to fluoroquinolone, specifically norfloxacin, are
sensitive to TPZ.
III.2.2 MRSA strains are sensitive to TPZ
Methicillin resistant S. aureus (MRSA) is one of the most difficult to treat
hospital infection. Both methicillin sensitive (MSSA) and resistant clinical isolates
of S. aureus were tested using drug spotting essay at various TPZ concentrations
under hypoxic condition [Figure 3.3]. The strains grew well in the absence of TPZ
[Figure 3.3ab]; however the growth inhibition by TPZ is evident at as low as 2μM
TPZ concentration [Figure 3.3cd]. There is a difference in viability among strain
isolates, though we do not have genotypic information on individual strain in
order to analyse them further. Interestingly, a slight increase in concentration to
3μM TPZ completely inhibits growth of both MSSA and MRSA [Figure 3.3ef]. This
experiment was also conducted under aerobic condition. S. aureus growth was
inhibited with 100μM TPZ (data not shown). In summary, MRSA strains are
41
sensitive to TPZ in both aerobic and anaerobic conditions, but the concentration
of TPZ required is 50 times lower in anaerobic condition.
III.2.3 C. difficile strains are sensitive to TPZ
C. difficile is a spore forming bacteria that is resistant to all the standard
disinfecting agents, which makes it more difficult to control its outbreaks in
hospitals. Eight potent strains of C. difficile were tested for sensitivity to TPZ by
drug spotting method described in CLSI manual [Figure 3.4]. Ampicillin was used
as a positive control. C. difficile strains are sensitive to TPZ at 1000 times lower
concentration compared to ampicillin. The MIC of TPZ for the eight strains tested
ranges from 7.5ng/ml to 30ng/ml. Nap2 strain is the most sensitive to TPZ
showing a growth defect at 3.75ng/ml and Nap5 strain is the least susceptible to
TPZ showing visible growth at 15ng/ml. Except Nap5, all strains are susceptible at
15ng/ml TPZ concentration.
III.3 TPZ is bactericidal
In order to understand the mechanism of action of TPZ, a time kill assay
was performed in both aerobic and anaerobic conditions. The shape of the graph
of time kill assay is indicative of whether the drug is bactericidal or bacteriostatic
[See I.1.2.3.2, Figure 1.4]. In this experiment, a rapid decline in viable cells was
observed when TPZ is added to growth medium compare the control bacterial
culture [Figure 3.5]. In aerobic condition (50μM TPZ), almost all cells were killed
after 4h under the influence of TPZ [Figure 3.5a]. There is an increase in growth
curve after 8h though it is still 106 times lower than the growth of the control
population.
In anaerobic condition (10μM TPZ), the shape of the killing curve is very
similar to aerobic condition [Figure 3.5b]. Although the growth defect of bacteria
via drug spotting assay was observed at 0.375μM [Figure 3.1b], a higher
concentration of 10μM TPZ was required in this experiment to observe a
significant difference in growth within a short period of time. Almost all cells
42
have been affected by TPZ at 8h, and again we observed difference of 106 CFU
after 24h between control media and TPZ containing media. As mentioned in
section I.1.2.3.2, bactericidal effect is highly dependent on the TPZ concentration
in the growth media. I also observed that the final CFUs of bacteria at 24h is
dependent on the starting concentration of TPZ. Nevertheless, the initial shape
of the curve remains intact, and the sharp decrease in growth between 4h to 8h,
encourages the conclusion that TPZ is bactericidal. Peculiarly, we see a small
peak in growth at approximately 6-7h into the experiment. Lower limit of
accuracy in these graphs is set at log 1 CFU/ml, as the number of viable cells can
be overestimated below this value.
III.4 Mutation in HR genes results in TPZ hypersensitivity
Homologous recombination (HR) is a process by which cell repairs DNA
damage. We tested all viable single gene knockout E. coli strains involved in HR
to confirm if DNA damage is the major reason for bactericidal activity of TPZ. This
experiment was only carried out under aerobic condition, because the specific
functionality of HR genes under anaerobic condition is unknown. Moreover, TPZ
is active in normoxia, albeit, not with the same potency as in the hypoxia. We
observe that WT strain is growing well, whereas recA, recB, recC, recF, recO and
recR mutants are hypersensitive to 7μM TPZ [Figure 3.6]. These genes are all
involved in HR. These results suggest that TPZ is most likely involved in direct or
indirect DNA damage. Other deletion strains recD, recN, and recJ were also
observed to be somewhat sensitive at 7μM TPZ and recQ was found to have
equal viability to that of WT (data not shown).
III.5 Bacterial reductase mutants are resistant to TPZ
In eukaryotes, TPZ is enzymatically activated mainly by cytochrome p450
reudctase. In order to understand the mode of action of TPZ, we focused on its
enzymatic activation in E. coli. Based on previous knowledge of human and yeast
cells, we decided to test functional gene knockouts of 197 putative reducatases
43
[Appendix A]. These were subjected to a wide range of TPZ concentrations under
aerobic and anaerobic conditions. The rationale behind this experiment was that
if a gene encoding reductase is involved in TPZ activation, then its deletion
should result in some form of resistance. Overall, we observed that there are
multiple reductases potentially involved in this pro-drug activation process. Gene
product ArgC shows the most amount of resistance at 10uM (normoxic
condition) and 0.375uM (hypoxic condition), which were determined as a cut off
concentrations because they are the closest tested concentrations at which
complete inhibition of wild-type growth is observed [Figure 3.7]. Therefore,
based on this criterion ArgC is nominated as the most important reductase for
TPZ activation both in normoxic and condition and placed at the top in Table 3.1.
YdhV and YeiA are observed to be more resistant than ArgC at 15uM (normoxic
condition) and 0.5uM (hypoxic condition), however these concentrations were
not chosen as the “cut-off” concentrations. It is highly plausible that more than
one reductase is involved in TPZ activation; therefore top ten most resistant
genes among all 197 have been tabulated in Table 3.1. In addition, we observed
that the involvement of reductases varies depending on the oxygen level during
incubation. Some reductases (e.g. YdhV and NapA) are more important in
aerobic condition, whereas RsxD and YeiA are more important in the absence of
oxygen. Other gene products such as YdbK, ProA, DmsB, NarG, and YjhC are less
involved in TPZ activation though show significant resistance [Table 3.1].
44
IV. DISCUSSION
IV.1 TPZ as a potential antibiotic
This thesis is raising a question whether TPZ can be used as an
antibacterial agent. In an effort to answer this question, I have gathered in vitro
evidences to support that TPZ may prove to be an effective antibiotic. I find that
E. coli lab strain, E. coli fluoroquinolone resistant strains, MRSA strains and C.
difficile strains are sensitive to TPZ at nM to μM concentrations. Since E. coli is a
gram-negative bacterium, and MRSA and C. difficile are gram-positive bacteria, I
infer that TPZ may prove to be a broad-range antibiotic. Also, I observed that TPZ
impose a similar sensitivity to all strains within that species be it WT strains or
MDR resistant strains of the same species. A MIC determination experiment
showed norfloxacin resistance similar to what has been reported in the literature
[243], however, all of these strains are highly sensitive to TPZ [Figure 3.2]. One of
the major limitations of MIC essay is that small amount of growth cannot be
detected visually. Optical density can be used as a quick way of quantifying
growth, although it may not be accurate at low values and values often
encompass turbidity of non-viable cells. A more advance method of viable cell
quantification via bacterial viability kits. These kits are based on a propidium
iodide dye which easily penetrates defective cell membrane of dead cells.
IV.1.1 C. difficile is sensitive to TPZ
C. difficile sensitivity to TPZ is higher than all reported antimicrobial agent
in Table 5 of CLSI document [248]. According to this database, the lowest MIC
reported for C. difficile strain ATCC 700057 (Strain #8 in Figure 3.4) is that of
Rifaximin between 3.9–15.6ng/ml. According to my observations, TPZ effects
Nap2 and Nap3 strains at as low as 1.8ng/ml [Figure 3.4]. TPZ also prevents
complete growth of ATCC 700057 strain at 15ng/ml or lower. Interestingly,
pathogenic strains like Nap1,2,3,4 are more sensitive to TPZ than WT ATCC
45
700057 strain. The variation in growth rate of each strain in hypoxia might affect
their sensitivity to TPZ. Nevertheless, MIC range of TPZ is still lower than
commonly used antibiotics for CDI such as vancomycin (0.5-4μg/ml), imipenem
(0.5-2μg/ml), metronidazole (0.125-0.5μg/ml) and newly introduced fidaxomicin
(0.06-0.25μg/ml)[248]. The MIC value for ampicillin is also reported previously
and it was easily available to be used as a positive control. Though ATCC 700057
strain was not sensitive at 4μg/ml concentration as reported, all strains were
sensitive to ampicillin at 10μg/ml. One of the main causes of CDI is intestinal
microbiota imbalance from exposure to broad range antibiotics. If TPZ was to be
a broad-spectrum drug, as it appears to be, we will have to take extra
precautions when introducing TPZ for CDI. TPZ is not selective for a specific
bacterial species, but it does require the lowest concentration to render C.
difficile sensitive compare to all other strains tested. Thus, if we are able to treat
CDI at low enough TPZ concentration, it may not affect other microbiota. Further
in vivo experiments and clinical trials are required to analyze this issue in detail.
Since vancomycin and metronidazole are no longer effective for CDI, there is an
urgent need for new antibiotic for C. difficile [249]. TPZ may fulfill this current
need for a highly potent antibiotic with novel mechanism of action.
IV.2 Mechanism of action of TPZ in bacteria
As described previously, WT and resistant strains are equally sensitive to
TPZ infers that TPZ may possess a distinct mechanism of action from that of
fluoroquinolone, methicilin, ampilicin, vancomycin and all other drugs which the
tested strains were resistant to. However, the ultimate effect of DNA damage in
eukaryotes may still be the same in prokaryotes. I have collected number of
evidences that supports the hypothesis that TPZ causes DNA damage in
prokaryotes. First of all, the growth curve of E. coli in medium containing TPZ
[Figure 3.5] matches closest to that of bactericidal growth curve [Figure 1.4]. In
order for a drug to be bactericidal, it must target a key cellular component like
46
DNA. Oddly, the curve gives a minor peak at 6h before reaching cell count to
negligible [Figure 3.5]. This minor peak might be a result of extreme survival
response of bacteria before lethality, decrease in TPZ potency over the time
period, increase production of non-toxic secondary metabolites of TPZ, or DNA
repair mechanisms in bacteria catching up to the DNA damage caused by TPZ.
The increase in population at 24h is attributable to the growth of small amount
of surviving cells dividing overnight. These cells might have developed resistance
to TPZ. I also conducted post antibiotic effect (PAE) experiment with TPZ, which
showed approximately 3-5h PAE after removing TPZ from the media (data not
shown). Indirect evidence that TPZ is involved in DNA damage is from
hypersensitivity of Rec mutants to TPZ [Figure 3.6]. In bacteria, Rec mutants play
a vital role in repairing DNA damage through HR. The functional absence of Rec
mutants decreases DNA repair capacity of the cell and eventually proves lethal.
Hypersensitivity of these mutants shows that TPZ is involved in causing DNA
breaks. The third evidence for DNA damage is that TPZ is positive in the Ames
test. Ames test is a commonly used technique to test if the given molecule is
mutagenic. I performed Ames test on TA100 Salmonella strain and the results
agree to those listed in TPZ MSDS sheet [96]. My attempt to collect direct
evidence of DNA damage, led me to perform in vitro experiments with
supercoiled plasmid DNA. I added TPZ and purified mammalian cytochrom P450
reductase enzyme to the mixture and observed nicks in supercoiled DNA by
upward mobility shift on agarose gel (data not shown). All of these results
combined indicate that TPZ is involved in direct or indirect DNA damage in
bacteria.
I have considered DNA gyrase as a target for TPZ and attempted multiple
in vivo and in vitro DNA gyrase assay with negative results (results not included).
Hence, DNA gyrase is unlikely to be the target in bacteria, though further
experiments are required to confirm these findings. TPZ shows potential as an
antibacterial agent in spite of the exact bacterial target being unknown. Since we
47
know that TPZ is DNA damaging, it may only be utilize for highly specific cases. It
should be used with caution for immune compromised patients; patients with
defect in DNA repair mechanisms for example, abnormal BRCA1/2 gene carriers.
(BRCA1/2 is involved in DNA repair mechanism and genetic mutations in these
are known to significantly increase risk of cancer [250].) So far the evidences
suggest that TPZ can be a valuable addition to the lineup of last resort antibiotics
to treat multi drug resistant pathogens.
IV.3 Hypoxia dependence of TPZ
We observe that it requires 25 times more TPZ for E. coli and almost 50
times more concentration of TPZ for S. aureus for it to be effective under aerobic
condition. (C. difficile cannot grow under aerobic condition; therefore this
difference cannot be measured for this organism.) This is expected owing to the
hypoxia mediated conversion of TPZ into its toxic species. The oxygen level in
human body varies significantly depending on the type of organ, tissue and
blood vessel. Although hypoxic conditions rarely occur in blood vessels of
healthy human body, tissues that are far from blood vessels are known to
maintain hypoxia [251, 252]. In addition, various inflamed and diseased tissues
are subjected to hypoxia or ischemia such as malignant tumor [158],
atherosclerosis [253], rheumatoid arthritis [254], wounds [255] and bacterial
infection [256, 257]. During bacterial infection, inflammation causes local
vasoconstriction of blood vessels and accumulation of phagocytes which can
hinder blood flow, and consequently oxygen availability. Furthermore, utilization
of oxygen by proliferating bacteria at the site of infection can cause local hypoxia
[258]. As a result, there is still potential for TPZ activation at the site of infection.
On the other hand, this very nature of TPZ increases its efficacy and specificity
towards infected tissues in the body by protecting uninfected and well
oxygenated cells. Also, specifically for pathogens like C. difficile which exclusively
grows in anaerobic environment, TPZ can prove to be highly effective. More than
48
TPZ’s reliance on hypoxic environment, the diagnostic challenges for hypoxia
detection can prove to be a bigger hurdle in TPZ treatment. In general HIF-1
expression is considered marker for hypoxia, however HIF-1 can be induced in
the absence of hypoxia by LPS and macrophages during bacterial infection [259,
260]. For this reason, one must be cautious as to make the correlation of hypoxic
microenvironment to HIF-1 expression particularly in the case of bacterial
infection.
IV.4 Resistance to TPZ
As we have witnessed throughout history, resistance is an inevitable
consequences for all antibiotic usage in clinical settings. There is no reason to
assume that TPZ will be resistance proof. Although the exact target in bacteria is
unknown, based on our current understanding in eukaryotic systems, we can
predict the means by which TPZ resistance can arise. First of all, bacterial
reductase mutants can cause resistance. Reductases are required to activate TPZ
to toxic compound. Therefore, if there is a mutation in reductase gene involved
in TPZ activation, those strains can turn resistant to TPZ. Due to multiple
reductases involved in TPZ activation, it is less likely that all of them acquired
mutation at the same time. If TPZ binds or utilizes DNA associated proteins in its
mechanism of toxicity, mutations in that protein associated gene sequence can
give rise to resistance. Previously, we discussed that TPZ causes DNA damage. As
a result, more mutations in DNA are likely to be formed upon TPZ usage, though
its bactericidal property can prevent them from accumulating and being passed
on to the next generation. I have performed two experiments through which I
have obtained TPZ resistant strains. First experiment was random chemical
mutagenesis using Ethyl methanesulfonate (EMS), through which I obtained
several resistant strains to TPZ under both aerobic and anaerobic conditions
(results not included). Though, I was unable to characterize the exact mutation
49
which led to resistance. The second set of TPZ resistant strains were obtained in
the form of reductase mutant strains [Table 3.1].
IV.4.1 Reductase mutants resistant to TPZ
In order to determine the reductase involved in TPZ activation, I
performed a screen of 197 E. coli strains carrying deletion of genes encoding
known or putative reductase or reductase-like functional gene knockouts. Many
of these deletion strains were resistant to TPZ. Table 3.1 summarizes the top 10
most involved reductases from my screen and I have decided to focus my
analysis on this particular subset. When BLAST search (filtered for E. coli K-12
strain) was carried out for the top hit, ArgC protein sequence, the result showed
only one of the 10 proteins, YjhC, with 44% identity. This suggests that there is
low sequence identity between these protein sequences. Next, I attempted
multiple sequence alignment of the 10 protein sequences but did not succeed in
finding a consensus sequence/region among them (data not shown). As a result,
I decided to use Conserved Domain Database (CDD) in order to analyze the 10
proteins [Table 5.1]. I observed that although each sequence is quite unique,
there are few common elements. For example, 50% of the listed (ArgC, YeiA,
RsxD, ProA and YjhC) proteins depend on NADP, NADH, or NADPH for their
activity. This is not surprising because NAD+ is a cofactor for reductases, and
there are some structural similarities between pyrimidine, NAD derivatives, and
TPZ such as benzene or triazine ring structure. Furthermore, several proteins are
involved in nitrogen related pathways including RsxD, NapA and NarG. NapA and
NarG both contain Molydopterin-binding super family domain and ArgC and YjhC
contain the common NADB_Rossmann super family domain.
It is noteworthy to observe that even though two proteins contain the
same domain; their activity against TPZ is not comparable. It is likely that other
factors apart from the oxidative domain can affect TPZ activation. Interestingly,
my results suggest that reductase activity is affected by oxygen levels during
growth on solid medium. Mutation in argC and ydhV creates highly resistant
50
mutants under aerobic condition, whereas, mutation in argC, yeiA and rsxD
creates highly resistant mutants under anaerobic condition. CDD analysis shows
that NapA, DmsB, and NarG are involved in anaerobic respiration; however, their
functional absence does not increase their resistance to TPZ. These strains
showed no growth defect under anaerobic condition, therefore the observed
phenotype is exclusively due to TPZ. Further experiments should be performed
with double or triple mutants of various genes from Table 3.1 (e.g. argC, yeiA
and ydhV mutation combined in a single strain) to observe the increase in
resistance to TPZ and further prove the involvement of these genes in TPZ
activation. In addition to inferring with TPZ mechanism, this study can also shed
light on functionality of poorly characterized proteins in this list, such as YeiA,
RsxD, YdhV, YdbK, and YjhC. They can be further investigated for their
involvement in drug metabolism in general.
V. FUTURE DIRECTIONS
All the experiments I have performed present preliminary evidence in
support of antibiotic use of TPZ. Albeit, experiments are performed only in
bacterial systems, to further confirm efficacy of TPZ as an antibiotic, in vivo
experiments with a mouse model system required. Primary experiment will
require approximately 18-20 adult mice. These can be infected with C. difficile
through a food source, or can be exposed to another broad spectrum drug which
indirectly leads to CDI in the large intestine. These mice can be divided into six
groups of three, where one group will be treated with clinically relevant dose of
vancomycin as a positive control. The other five groups will be exposed to five
different concentrations of TPZ ranging from 30ng/ml to 1ug/ml. The mice
should be monitored for toxicity and recovery time from infection compared to
vancomycin.
To decrease the toxicity of TPZ to humans, several structural
modifications can be done, however one needs to be careful not to decrease its
51
potency in the process. I propose testing numerous derivatives of TPZ already
available in the literature [Table 1.2] first before investing resources in designing
new molecules. These TPZ analogues and derivatives should be screened for
their efficacy against various pathogens first in vitro, followed by in vivo analysis
of promising compounds.
There also needs to be further investigation on a specific target of TPZ.
DNA damage is the ultimate effect of TPZ toxicity; however whether it is caused
directly or indirectly is unknown. In the bacterial system, DNA is not
compartmentalized in a nucleus, therefore in theory all cellular proteins can
come in contact with TPZ. This makes the task of target hunting highly intricate.
If any protein is involved in TPZ mechanism in bacteria, involvement of DNA
associated proteins (in addition to DNA gyrase) should be of primary suspect.
Once the proteins involved are narrowed down, computerized molecular
simulations can be done to confer potential binding site of TPZ on these
proteins. Once narrowed down further, protein gene can be cloned on a plasmid
and expressed artificially in E. coli. Such genetic experiments can be performed
with functional knockout of associated genes to see their effect on TPZ
sensitivity, followed by substituting the cell with the gene plasmid that can
rescue the original phenotype. DNA associated proteins are often essential genes
for which mutants cannot be obtained. If this is the case, the protein can be over
expressed to see its effect on TPZ sensitivity. If the target is found, x-ray
crystallography can provide further structural insights. The second challenge in
target identification is that it is difficult to experiment with short lived, radical
form, active TPZ species. Moreover, the requirement of hypoxia for optimal
function of TPZ is an added challenge in performing target defining experiments.
Whole genome sequencing of TPZ resistant strains can also give some insights
into the target genes involved in TPZ mechanism.
Clinically, there needs to be further studies on tolerable dozes of TPZ,
especially as an antibiotic. Various side effects seen previously may disappear if
52
TPZ can act as an antibiotic at a much lower dosage. Supplementary research in
detection methodologies of precise hypoxia markers in the cell and the role of
hypoxia in bacterial infection can significantly impact utility of hypoxia activated
drugs.
V.1 Conclusion
Through this thesis I have researched alternative use of a well-studied
anti-cancer drug TPZ. I have collected promising results on effectiveness of TPZ in
both gram-negative and gram-positive pathogens that are resistant to majority
of existing antibiotics in the market. My results show that rigorous pathogens
like MRSA and C. difficile are sensitive to TPZ at low concentrations. I find that
TPZ has bactericidal effect in E. coli, and is involved in cellular DNA damage.
There are evidences to support that TPZ might have similar mechanism to that of
eukaryotes via resistance of reductase mutants to TPZ. In addition to providing
an antibiotic with novel mechanism, TPZ has the potential to cure last-stage
hospital infections which are currently challenging to treat. Providing that TPZ is
already well-studied and in phase III clinical trials for cancer, it requires lower
investment for companies willing to take this drug to the market. Just because
major pharmaceutical companies have discontinued their interest in antibiotic
development, emergence of multi-drug resistance bacteria will not halt. There is
and will always be pressing need for new molecules that can combat pathogenic
microbes.
53
VI. FIGURES
Figure 1.1: The perfect storm. Increasing cases of MDR infections coincides with a steady decline in research and development of new antibiotics. Taken from reference [261].
Figure 1.2: Drying pipeline of antibiotics. A historical timeline of introduction of new classes of antibiotic drugs. There have only been 5 new classes of drugs introduced since 1968 compared to 14 classes of antibiotics prior to that. Taken from reference [262].
54
Figure 1.3: Structural formula of various antibiotics A) Penicillin B) Vancomycin C) Novobiocin D) Ciprofloxacin E) Levofloxacin F) Norfloxacin.
A
B
C
D
E
F
55
Figure 1.4: Logarithmic growth curve of bacteria under influence of bacteriostatic versus bactericidal drug determined by counting viable colonies at various time points. Taken from reference [18].
Figure 1.5: Drug resistant mechanisms. A schematic outlining major
mechanisms by which bacteria acquire resistance to antibiotic. Taken from reference [43].
56
Figure 1.6: Chemical structure of Tirapazamine [130]
Figure 1.7: General mechanism of preferential cytotoxicity of TPZ. Taken from reference [138].
57
Figure 1.8: A schematic of DNA gyrase (a topoisomerase II). DNA gyrase acts on positively supercoiled DNA to change its linking number by 2. Taken from reference [263].
Figure 1.9: C. difficile under microscope [264] (left). C. difficile growing on blood agar plate [265] (right).
58
Figure 3.1:
0.0
00
0.1
25
0.2
50
0.3
75
+O2
0.0
6.5
8.0
10
.0
TPZ(μM)
TPZ(μM)
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Fig
ure
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59
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60
Figure 3.3: S. aureus strains are sensitive to TPZ under low O2 levels. A, B) All strains grow normally in the hypoxic environment when no TPZ is added to the growth media. C, D) The growth defect is visible in some strain at 2μM TPZ. E, F) A low concentration of 3μM TPZ completely inhibits growth of both MSSA and MRSA. Plates were incubated overnight at 37°C under anaerobic conditions. The most concentrated spot on the plate is diluted by factor of 0.01 from overnight growth. Methicillin-sensitive Staphylococcus Aureus (MSSA) strains are: MA076688, MA076723, MA076899, MA077045. Methicillin-resistant Staphylococcus aureus (MRSA) strains are: MA077046, MA077064, MA077074, and MA077085. The final plating volume is 5μl.
TPZ(μM) 0 2 3
MR
SAM
SSA
-O2
A
B
C
D
E
F
61
Figure 3.4: C. difficile is sensitive to TPZ. The strains indicated by the numbers are as follows 1. Nap1 2. Nap2 3. Nap3 4. Nap4 5. Nap5 6. Nap6 7. CIP 107932 8. ATCC 700057. The MIC is in the range of 7.5-30ng/ml TPZ. Ampicillin (Amp) was used as a positive control. This experiment was carried out under anaerobic condition. The plating volume is approximately 2μl.
1 2 3 4 5 6 7 8
1.80
3.75
7.50
15.0
30.0
TP
Z(n
g/m
l)A
mp
(μg
/ml) 4
10
Control
62
Figure 3.5: TPZ is bactericidal under both aerobic and anaerobic conditions. Log of CFU count per ml of culture for each time point is plotted on the y-axis. There was no TPZ added to control culture at any time point (top curve), however, 50uM (A) and 10uM (B) TPZ was added to growth media in an otherwise identical sample at 0h (bottom curve). A) Time-kill curves under aerobic condition performed in a 500ml flask at 37°C. B) Time-kill curves under low oxygen condition performed in 15ml culture tubes in an anaerobic chamber (jar) at 37°C. The cells were plated on LB plates and incubated overnight before manual count of CFUs. The plotted CFU value is an average of three dilutions plated for each time point. BW25113 strain of E. coli was used for this experiment.
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12 14 16 18 20 22 24 26
log
CFU
/ml
Aerobic incubation time (h)
Control
50μM TPZ
Lower limitof accuracy
AA
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12 14 16 18 20 22 24 26
log
CFU
/ml
Anaerobic incubation time (h)
Control
10μM TPZ
Lower limitof accuracy
B
63
Figure 3.6: TPZ is likely to be involved in direct or indirect DNA damage. Strains lacking genes involved in homologous recombination are hypersensitive to TPZ at 7uM concentration. Strains were obtained from the Keio collection [Appendix A2] and the experiment was carried out under aerobic condition. LB plates with and without 7μM TPZ were used and the platting volume was 5μl.
Figure 3.7: Functional deletion of putative reductases increase resistance to TPZ in E. coli. Four out of ten such mutant phenotypes are presented in this figure. Growth score for the rest have been tabulated in Table 3.1. Out of 197 gene knockouts used in the screen, the most interesting 25 knockouts were screened repeatedly for 3-6 times on various TPZ concentrations. WT stands for BW25113 E. coli strain. LB plates were used with or without TPZ and the platting volume was 10μl (see the next page).
WT
∆recA
∆ recB
∆ recC
∆ recF
∆ recO
∆ recR
+O2
TPZ(μM) 0 7
64
Figure 3.7 (see the previous page for caption)
WT
∆a
rgC
∆rs
xD
∆y
dh
V
∆y
eiA
TP
Z(μ
M)
0
0.2
50
.37
5
0.5
-O2
WT
∆a
rgC
∆rs
xD
∆y
dh
V
∆y
eiA
TP
Z(μ
M)
0
81
0
15
+O
2
65
VII. TABLES
Table 1.1
Summary of major antibiotics, their target proteins and
dominating resistance mechanisms. [Taken from reference
[17], table 2.5]
Classified Group
Antibiotics Target Resistance
Mechanism
Cell Wall
B-lactams Transpeptidases/transg
lycosylases(PBPs*)
B-lactamases, PBP
mutants
Vancomycin,
Teicoplanin
D-Ala-D-Ala termini of
peptidoglycan and of
lipid II
Reprograming of D-
Ala-D-Ala to D-Ala-D-
Lac or D-Ala-D-Ser
Protein
Synthesis
Erythromycins Peptidyltransferase/rib
osome
rRNA
methylation/efflux
Tetracyclines Peptidyltransferase Drug efflux
Aminoglycosides Peptidyltransferase Drug modification
Oxazolidinones Peptidyltransferase Unknown
DNA
replicatio
n/repair
fluoroquinolones DNA gyrase Gyrase mutations
*PBP= Penicillin Binding Protein
66
Table 1.2
A representative subset of structural modifications attempted
on TPZ in order to increase its clinical potential as an
anticancer drug.
Modifications on TPZ (Refer to Fig. 1
numbering of atoms)
Number of compounds synthesized/
tested
Major observations (Compared to TPZ)
Reference
Methoxy group on 7-position, and Aromatic
group at 3-position
18 derivatives
3 compounds showed improved cytotoxicity
and selectivity [266]
SR-PKPD* guided lead optimization protocol
281 analogues
2 promising hypoxic cell toxins
[267]
Thienyl group at 2-position
10 analogues
One of them was tested extensively for its DNA
strand cleaving properties
[268, 269]
Small lipophilic groups into the C3-amino group, and electron donating or adopting substituents at
the benzene ring
22 derivatives
2 compounds show more cytotoxicity and hypoxic selectivity in
various cell lines.
[270]
3-alkylamino homologues at various amine pKa
43 analogues
Improved extravascular transport
[119, 271]
Added alkyl and aromatic groups into C3-amino
group in hopes of improving lipophilicity.
15 derivatives
2-3 compounds showed more potent activity in Molt-4 and HL-60 cell
lines
[272]
DNA-affinic chromophore linker attached into C3-
amino group.
11 analogues
7 compounds showed increase in cytotoxicity
while retaining selectivity to hypoxic cells.
[273]
3-Alkyl derivatives (8), 3-N,N-
dialkylaminoalkylamino derivatives (11), 3-amino
derivatives (4), and 3- desamino derivatives(2)
25 analogues
2 comounds (3-ethyl, and 3-propyl analogues)
showed improved aqueous solubility while other parameters remain
comparable to TPZ.
[274]
*SR-PKPD= spatially resolved pharmacokinetic/pharmacodynamic
model
67
Table 3.1
List of putative reductase genes, deletion of which results in
TPZ resistant phenotype in the screen of 197 deletion strains.
Keio
Collection
ID
Gene locus 10μM TPZ*
( +O2)
0.375μM
TPZ* (-
O2)
BW25113 WT −−−− −−−−
JW3930 argC ++++ ++++
JW2134 yeiA ++−− ++++
JW1622 rsxD ++−− +++−
JW5272 ydhV +++− ++−−
JW2194 napA +++− ++−−
JW0233 proA ++−− ++−−
JW1372 ydbK +−−− ++−−
JW0878 dmsB ++−− +−−−
JW1215 narG +−−− +−−−
JW5769 yjhC ++−− −−−−
*Cut off concentration at which growth of the wild-type strain is
completely inhibited. “+” indicates visible growth and “-” indicates
complete absence of growth. JW ID= Genobase ID. WT= wild-type.
68
Table 5.1
Bioinformatics analysis of putative bacterial reductases that
may activate TPZ.
Gene ID
Protein Ref
Gene Name
Name (Associated with NCBI Protein Ref)
Conserved Domain Database (CDD) results*
Predicted function of Conserved domains
16131796
NP_418393.1
argC / Arg2/argH (EC 1.2.1.38)
N-acetyl-gamma-glutamylphosphate reductase
NADB _Rossmann super family and Glyceraldehyde 3-phosphate dehydrogenase C-terminal domain
NAD(P)-binding.Involved in Arginine biosynthesis,”reference”cytoplasmic location,
90111398
NP_416652.4
yeiA Dihydropyrimidine dehydrogenase (DHPD), NADH-dependent, subunit B
DHPD_FMN domain which contains two FAD, two FMN and eight [4Fe-4S] clusters
NADH dependent reduction of Uracil and Thymine
16129588
NP_416147.1
rsxD electron transport complex protein required for the reduction of SoxR
NQR2_RnfD_RnfE super family, predicted membrane protein
sodium-translocating NADH-ubiquinone oxidoreductase and Nitrogen fixating proteins
16129629
NP_41618
ydhV Predicted oxidoreductase
AFOR_N super family and AFOR_C
Aldehyde ferredoxin oxidoreductas
69
8.1 super family e (AFOR) catalyzes reversible oxidation of aldehydes.
16130143
NP_416710.1
napA/yojC/yojD/yojE (EC 1.7.99.4)
nitrate reductase, periplasmic, large subunit
Molydopterin-binding super family (MopB) and MopB_CT super family
anaerobic respiration; Reduction of nitrate to nitrite
16128229
NP_414778.1
proA (EC 1.2.1.41)
γ- glutamylphosphate reductase (GPR)
ALDH-SF super family
Proline Biosynthesis; NADPH dependent reduction of L-gamma-glutamyl 5-phosphate into L-glutamate 5-semialdehyde and phosphate
16129339
NP_415896.1
ydbK/nifJ
pyruvate-flavodoxin oxidoreductase (PFOR or POR) conserved protein/FeS binding protein
PYR super family, POR super family, Fer4 super family and TPP_enzymes super family
Conserved protein, Pyrimidine(PYR) binding domain, Thiamine pyrophosphate(TPP) dependent enzyme
16128862
NP_415415.1
dmsB (EC 1.8.99.-)
dimethyl sulfoxide reductase, anaerobic,
Fer4 super family and DMSO_dmsB
Aneribic respiration; attached to inner membrane via
70
subunit B
chain C. Chain A and B are catalytic. Binds to Iron sulfer clusters
GI:16129187
NP_415742.1
narG /narC/bisD/chlC (EC 1.7.99.4)
nitrate reductase 1, alpha subunit
Molydopterin-binding super family (MopB) and MopB_CT super family
Anaerobic respiration, inner membrane bound, NarGHI (quinol-nitrate oxidoreductase) reduces nitrate to nitrite and contributes in generating proton motive force.
90111719
NP_418700.2
yjhC KpLE2 phage-like element; predicted oxidoreductase
NADB_Rossmann super family and GFO_IDH_MocA_C super family
Contains NAD(P)/NADP(+) binding domain, the second domain can utilize NADP or NAD.
* Domain analysis from Conserved Domain search of protein sequences in FASTA format (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
71
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88
IX. APPENDIX
Appendix A
Table A: List of genes tested for sensitivity to TPZ. They are either
putative reductases, predicted reductases, or chosen from blast hits of
human p450 reductase.
JW ID EckID* Gene and other names
GeneID EckID Gene and other names
GeneID EckID Gene and other names
JW2452 ECK2463 aegA/yffG JW1463 ECK1462 narZ/chlZ JW0752 ECK0758 ybhH
JW0598 ECK0599 ahpC/tpx JW1095 ECK1095 ndh JW0837 ECK0844 ybjN
JW0599 ECK0600 ahpF JW1642 ECK1646 nemA/ydhN JW5819 ECK0859 ybjS
JW3930 ECK3949 argC/Arg2/argH
JW0567 ECK0570 nfnB/nfsI/nfsB/ntr/dprA
JW5126 ECK0938 ycbx
JW3242 ECK3268 aroE JW0835 ECK0842 nfsA/mdaA/mda18/ybjB
JW0952 ECK0960 yccK
JW3470 ECK3488 arsC/arsG JW3328 ECK3353 nirB JW5138 ECK0998 ycdH
JW1409 ECK1405 azoR/acpD JW3329 ECK3354 nirD JW0993 ECK0999 ycdI
JW5940 ECK3538 bisC JW2680 ECK2705 norV/flrD/ygaK/flrd/ygaI/ygaJ
JW5146
ECK1019 ycdW
JW0038 ECK0040 caiA/yaaO JW2681 ECK2706 norW/flrR/ygbD/ygaL
JW1263 ECK1265 yciK
JW5631 ECK3682 cbrA/yidS JW4197 ECK4233 nrdD JW1258 ECK1260 yciv
JW2732 ECK2757 cysH JW2650 ECK2669 nrdE JW5200 ECK1282 yciW
JW2733 ECK2758 cysI/cysQ JW2651 ECK2670 nrdF/ygaD JW1306 ECK1308 ycjQ
JW2734 ECK2759 cysJ/cysP JW4196 ECK4232 nrdG/yjgE JW1308 ECK1310 ycjS
JW5499 ECK3004 dkgA/yqhE JW4031 ECK4063 nrfA/aidC JW1403 ECK1399 ydbC
JW0197 ECK0207 dkgB/yafB JW4032 ECK4064 nrfB/yjcI JW1403 ECK1399 ydbc
JW5118 ECK0885 dmsA JW4033 ECK4065 nrfC/yjcJ JW1372 ECK1374 ydbK/nifJ
JW0878 ECK0886 dmsB JW4034 ECK4066 nrfD/yjcK JW1495 ECK1494 ydeP
JW0879 ECK0887 dmsC JW2283 ECK2282 nuoA JW5265 ECK1619 ydgJ
JW5182 ECK1173 dsbB/roxB/iarB/dsbX/ycgA
JW5875 ECK2281 nuoB JW1639 ECK1643 ydhF
JW3052 ECK3071 fadH/ygjL JW5375 ECK2280 nuoC/nuoCD/nuoD
JW5272 ECK1669 ydhV
JW2037 ECK2046 fcl/wcaG/yefB JW2280 ECK2279 nuoE JW1677 ECK1684 ydiJ
JW4331 ECK4357 fhuF/yjjS JW2279 ECK2278 nuoF/ygfQ/nuoB
JW1689 ECK1697 ydiS
JW0042 ECK0044 fixC/yaaS JW2278 ECK2277 nuoG JW1754 ECK1763 ydjA
JW0671 ECK0672 flda JW2277 ECK2276 nuoH JW1760 ECK1769 ydjG
89
JW1598 ECK1601 folM/ydgB JW2276 ECK2275 nuoI JW1763 ECK1772 ydjJ
JW3895 ECK3916 fpr/mvrA/flxR JW2275 ECK2274 nuoJ JW1765 ECK1774 ydjL
JW4115 ECK4150 frdA JW2274 ECK2273 nuoK JW1767 ECK1776 yeaA/msrB
JW4114 ECK4149 frdB JW2273 ECK2272 nuoL JW1770 ECK1779 yeaE
JW4113 ECK4148 frdC JW2272 ECK2271 nuoM/nuoA JW1792 ECK1801 yeaX
JW4112 ECK4147 frdD JW2271 ECK2270 nuoN JW1954 ECK1967 yedY
JW3820 ECK3836 fre/flrD/fadI/fsrC/ubiB/luxG
JW1383 ECK1385 paaA/ydbO JW2134 ECK2140 yeiA
JW2770 ECK2794 fucO JW1384 ECK1386 paaB/ynbF JW2133 ECK2139 yeiT
JW5526 ECK3113 garR/yhaE JW1385 ECK1387 paaC/ydbP JW2230 ECK2228 yfaE
JW0497 ECK0502 glxR/glxB1ybbQ/ybbQ/glxB1
JW5217 ECK1388 paaD/ydbQ JW2338 ECK2335 yfcX/fadJ
JW2038 ECK2047 gmd/yefA/yefN JW1387 ECK1389 paaE/ydbR JW2480 ECK2491 yfgD
JW3467 ECK3485 gor/gorA JW0415 ECK0419 panE/apbA JW2736 ECK2761 ygcN
JW0833 ECK0840 grxA/grx JW0389 ECK0393 phoB/phoRc/phoT
JW2870 ECK2897 ygfF/yqfD
JW0101 ECK0104 guaC JW0233 ECK0244 proA/proL/pro1/pro(1)
JW5923 ECK2874 ygfK
JW2526 ECK2539 hcaD/phdA/hcaA4/yfhY/hcaa4
JW0377 ECK0381 proC/Pro2/pro3/pro(3)
JW2848 ECK2876 ygfM
JW0857 ECK0864 hcp/ybjW/priS JW1293 ECK1295 puuC/aldH JW5468 ECK2881 ygfS
JW5117 ECK0863 hcr/ybjV JW4011 ECK4043 qor/hcz/hzc JW5469 ECK2882 ygfT
JW2536 ECK2549 hmp/hmpA/fsrB
JW2025 ECK2034 rfbD/rmlD/rfbC JW2922 ECK2950 yggW
JW3733 ECK3748 HsrA/yieO JW3765 ECK3783 rffA/wecE/yifI/fcnA
JW2970 ECK2995 yghZ/mgrA
JW4223 ECK4259 idnO/yjgU JW1572 ECK1575 rspB JW3178 ECK3201 yhcC
JW2447 ECK2458 maeB/ypfF JW1622 ECK1626 rsxD/rnfD/ydgO
JW3222 ECK3241 yhdH
JW2996 ECK3019 mdaB/mda66 JW1624 ECK1628 rsxE/rnfE/ydgQ
JW3403 ECK3425 yhhX
JW4080 ECK4112 melA/mel-7 JW1623 ECK1627 rsxG/rnfG/ydgP
JW3459 ECK3477 yhiN
JW3913 ECK3933 metF JW0920 ECK0928 ssuE/ycbE/ycbP/ssi4
JW3691 ECK3706 yieF
JW2671 ECK2696 mltB JW2802 ECK2830 tas/ygdS JW3853 ECK3875 yihU
JW3720 ECK3736 mioC/yieB JW5656 ECK3540 tiaE/tkrA/yiaE/ghrB
JW4207 ECK4243 yjgI
JW4178 ECK4215 msrA/pms/pmsR
JW0982 ECK0988 torA JW5769 ECK4270 yjhC
JW1055 ECK1053 mviM/yceM JW0981 ECK0987 torC JW5793 ECK4348 yjjN
JW2194 ECK2198 napA/yojC/yojD/yojE
JW0983 ECK0989 torD JW5040 ECK0303 ykgC
JW5367 ECK2195 napB/yejY JW1862 ECK1874 torY/yecK JW5041 ECK0305 ykgE
JW2190 ECK2194 napC/yejX JW1861 ECK1873 torZ/bisZ JW5960 ECK1323 ymjC
90
JW2195 ECK2199 napD/yojF JW0871 ECK0879 trxB JW5907 ECK1443 yncB
JW2196 ECK2200 napF/yojG JW1514 ECK1514 uxaB JW1579 ECK1582 ynfE
JW2193 ECK2197 napG/yojA/yojB JW4286 ECK4314 uxuB JW5260 ECK1583 ynfF
JW2192 ECK2196 napH/yejZ JW2874 ECK2901 visC JW1581 ECK1584 ynfG
JW1215 ECK1218 narG/narC/bisD/chlC
JW0278 ECK0283 yagR JW5261 ECK1585 ynfH
JW1216 ECK1219 narH/chlC JW0279 ECK0284 yagS JW2125 ECK2130 yohF/yohE
JW1218 ECK1221 narI/chlI JW0317 ECK0323 yahK JW5842 ECK2542 yphC
JW1217 ECK1220 narJ JW0409 ECK0413 yajO JW2761 ECK2784 yqcA
JW1460 ECK1459 narV/chlZ JW0482 ECK0487 ybbO JW2867 ECK2894 yqfA
JW1461 ECK1460 narW/chlZ JW0592 ECK0593 ybdH JW3163 ECK3185 yrbG
JW1462 ECK1461 narY/chlZ JW0601 ECK0602 ybdR JW4169 ECK2463 ytfG
Appendix A2
Table A2: List of homologous recombination mutants tested for TPZ
sensitivity under aerobic condition.
JW ID EckID Gene Name Description
JW2669 ECK2694
recA/lexB/umuB/recH/rnmB/tif/zab/srf/umuR
DNA strand exchange and recombination protein with protease and nuclease activity
JW2788 ECK2816 recB/rorA/ior exonuclease V (RecBCD complex), beta subunit
JW2790 ECK2818 recC exonuclease V (RecBCD complex), gamma
chain JW2787 ECK2815 recD/hopE exonuclease V (RecBCD complex), alpha chain
JW3677 ECK3692 recF/uvrF Gap repair protein
JW2860 ECK2887 recJ ssDNA exonuclease, 5' --> 3'-specific
JW5416 ECK2612 recN/radB recombination and repair protein
JW2549 ECK2563 recO Gap repair protein
JW5855 ECK3816 recQ ATP-dependent DNA helicase
JW0461 ECK0466 recR Gap repair protein
91
Appendix B
Sample calculation of MIC ratio
For example, G81C strain in TPZ growth medium.
MIC WT = 17uM
MIC Mutant = 8uM
MIC ratio= MIC mutant/MIC WT
= 8uM /17uM
= 0.47
Average Ratio = (0.47 + 0.53 + 0.67) /3 = 0.56
Standard Deviation = x
; where x = data value, x = mean average of
data, and n = number of data points
=
= 0.10
Table B: Raw data from MIC determination experiment for growth of
gyrase mutant strains with TPZ.
Strains
Exp1 MIC (uM)
MIC Ratio 1
Exp2 MIC (uM)
MIC Ratio 2
Exp3 MIC (uM)
MIC Ratio 3
Ave Ratio
Std. Deviation
WT 17 10 12
S83L 17 1.00 10 1.00 12 1.00 1.00 0.00
A84P 20 1.17 8 0.80 12 1.00 0.99 0.19
D87Y 25 1.47 12 1.20 12.5/15
* 1.25 1.30 0.14
S83W 15 0.88 10 1.00 10 0.83 0.91 0.08
A67S 17 1.00 12 1.20 15 1.25 1.15 0.13
D87N 17 1.00 12 1.20 15 1.25 1.15 0.13
G81C 8 0.47 8/15* 0.53 10/15* 0.67 0.56 0.10
Q106H
17 1.00 10 1.00 15 1.25 1.08 0.14
D426N
15 0.88 10/15* 0.67 8 0.67 0.74 0.12
* Experiments were performed independently where WT MIC was 15 and mutant MIC is as indicated.
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