investigations of the natural product antibiotic thiostrepton from streptomyces azureus and ...

274 Pages·2013·14.17 MB·English

I
NVESTIGATIONS OF THE NATURAL
PRODUCT ANTIBIOTIC THIOSTREPTON
S
FROM TREPTOMYCES AZUREUS AND

ASSOCIATED MECHANISMS OF RESISTANCE

by

Cullen Lucan Myers
A thesis
presented to the University of Waterloo
in fulfillment of the
thesis requirement for the degree of
Doctor of Philosophy
in
Chemistry

Waterloo, Ontario, Canada, 2013

© Cullen Lucan Myers 2013
AUTHOR’S DECLARATION
I hereby declare that I am the sole author of this thesis. This is a true copy of the
thesis, including any required final revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

ii
ABSTRACT
The persistence and propagation of bacterial antibiotic resistance
presents significant challenges to the treatment of drug resistant bacteria with
current antimicrobial chemotherapies, while a dearth in replacements for these
drugs persists. The thiopeptide family of antibiotics may represent a potential
source for new drugs and thiostrepton, the prototypical member of this antibiotic
class, is the primary subject under study in this thesis.
Using a facile semi-synthetic approach novel, regioselectively-modified
thiostrepton derivatives with improved aqueous solubility were prepared. In vivo
assessments found these derivatives to retain significant antibacterial ability
which was determined by cell free assays to be due to the inhibition of protein
synthesis. Moreover, structure-function studies for these derivatives highlighted
structural elements of the thiostrepton molecule that are important for
antibacterial activity.
Organisms that produce thiostrepton become insensitive to the antibiotic
by producing a resistance enzyme that transfers a methyl group from the co-
factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the
thiostrepton binding site on 23S rRNA, thus preventing binding of the
antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to
generate point mutations at key active site residues. Ensuing biochemical assays
and co-factor binding studies on these variants identified amino acid residues in
the active site that are essential to the formation of the AdoMet binding pocket
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and provided direct evidence for the involvement of an active site arginine in
the catalytic mechanism of the enzyme.
Certain bacteria that produce neither thiostrepton nor the resistance
methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-
AS, that irreversibly bind to the antibiotic, thereby conferring resistance by
sequestration. Here, it was found that the point mutation of the previously
identified reactive amino acid in TIP-AS did not affect covalent binding to the
antibiotic, which was immediately suggestive of a specific, high affinity non-
covalent interaction. This was confirmed in binding studies using chemically
synthesized thiostrepton derivatives. These studies further revealed structural
features from thiostrepton important in this non-covalent interaction. Together,
these results indicate that thiostrepton binding by TIP-AS begins with a specific
non-covalent interaction, which is necessary to properly orient the thiostrepton
molecule for covalent binding to the protein.
Finally, the synthesis of a novel AdoMet analogue is reported. The
methyl group of AdoMet was successfully replaced with a trifluoromethyl
ketone moiety, however, the hydrated form (germinal diol) of this compound
was found to predominate in solution. Nevertheless, the transfer of this
trifluoroketone/ trifluoropropane diol group was demonstrated with the
thiopurine methyltransferase.

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ACKNOWLEDGEMENTS
My sincerest thanks are expressed to my supervisor Professor John
Honek, for his steady tutelage, guidance and support throughout my doctoral
studies. I am deeply grateful for his fostering the growth of my self-confidence
as a researcher, by encouraging open discussions about ideas and his unfailing
way of steering me in a right direction without belittlement and always ensuring
that I was learning in the process.
I also wish to thank the members of my committee, Drs. Dieckman,
Guillemette and Wettig for their constructive advice which helped to advance
my project.
My gratitude extends to the many members of the Honek Laboratory
over the years that provided their friendship and support, foremost among whom
is Dr.Pei Hang, who began the work on this project. I am immensely grateful for
her spending the time to train me (in spite of being in the process of preparing
her own doctoral thesis). I would also especially like to thank Drs. Zhengding
Su, Uthiawan Suttisansanee and Betsey Daub for sharing scientific expertise
and advice that was tremendously helpful at many different stages of my
studies. Thanks are also owed Joshua Yuen, Jesse Harris and Jacky Yeung, who
assisted in the synthesis of thiostrepton analogues and in studies of thiostrepton
resistance proteins.
I am deeply grateful to the Department of Chemistry at the University of
Waterloo for wonderful technical support that was vital to the success and
understanding of many aspects of this project. In particular, I would like to
thank Dr. Richard Smith for mass spectrometry training, invaluable advice and
suggestions; Jan Venne for NMR training and assistance with two-dimensional
NMR experiments; and Valarie Goodfellow and the Dmitrienko group for
allowing me access to their HPLC equipment. I am additionally grateful to all
the administrative and support staff of the Department of Chemistry. As well, I
greatly appreciate the gracious assistance of Marcie Chaudet and the Rose group
(Department of Biology) with X-ray crystallography.
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Finally, I would like to express my deepest and heartfelt gratitude to my
family for their unwavering support, love and understanding, which has been a
great source of motivation and inspiration throughout my studies. Thank you, all
of you, for your encouragement during my times of despair.

For Mama & Janine
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TABLE OF CONTENTS
LIST OF TABLES ............................................................................ xiii
LIST OF FIGURES ........................................................................... xv
LIST OF ABBREVIATIONS .............................................................. xix

CHAPTER 1: INTRODUCTION ........................................................ 1
1.1. Antibiotic action and mechanisms of resistance ................................................... 2
1.1.1. Target sites and modes of action for common antibiotics ............................ 4
1.1.1.1. Cell wall synthesis and cell membrane disruption ............................. 4
1.1.1.1.1. Inhibitors of peptidogylcan synthesis ....................................... 4
1.1.1.1.2. Bacterial cell membrane .......................................................... 5
1.1.1.2. Ribosome targeting antibiotics ........................................................... 5
1.1.1.3. Antibiotics targeting DNA synthesis and replication ......................... 10
1.1.2. Mechanisms of antibiotic resistance ............................................................. 11
1.1.2.1. Drug efflux ......................................................................................... 11
1.1.2.2. Antibiotic modification ...................................................................... 12
1.1.2.3. Target site modification...................................................................... 13
1.1.2.4. Molecular neutralization ..................................................................... 13
1.1.3. Emergence and transfer of antibiotic resistance ........................................... 14
1.1.4. Antibiotic development ................................................................................ 17
1.2. Thiostrepton and the thiopeptide family of antibiotics ......................................... 18
1.2.1. Structure and physical properties ................................................................. 18
1.2.2. Total synthesis and biosynthesis of thiostrepton .......................................... 21
1.2.3. Biological properties of thiostrepton ............................................................ 26
1.2.3.1. Antibacterial activity .......................................................................... 26
1.2.3.2. Additional biological properties of thiostrepton ................................. 30
1.3. Thiostrepton resistance ......................................................................................... 31
1.3.1. Resistance in thiostrepton producing organisms .......................................... 31
1.3.1.1. RNA methylation and antibiotic resistance ........................................ 34
1.3.1.2. AdoMet dependent methyltransferases .............................................. 36
1.3.1.3. Mechanism of transmethylation ......................................................... 37
1.3.1.4. AdoMet-dependent methyltransferases as tools ................................. 39
1.3.2. Resistance in organisms that do not produce thiostrepton ............................ 43
1.3.2.1. Thiostrepton resistant mutants ............................................................ 43
1.3.2.2. Thiostrepton binding proteins ............................................................ 45
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1.4. Research objectives ............................................................................................... 47

CHAPTER 2: SYNTHESIS AND BIOLOGICAL ACTIVITY OF
THIOSTREPTON ANALOGUES
2.1. Preface .................................................................................................................. 49
2.2. Materials and methods .......................................................................................... 51
2.2.1. Chemicals and reagents ................................................................................ 51
2.2.2. Equipment .................................................................................................... 52
2.2.2.1. Chromatography ................................................................................. 52
2.2.2.2. Mass spectrometry .............................................................................. 52
2.2.2.3. Luminescence ..................................................................................... 52
2.2.2.4. Nuclear magnetic resonance ............................................................... 52
2.2.3. General methods ........................................................................................... 53
2.2.3.1. Synthesis of thiostrepton analogues ................................................... 53
2.2.3.2. Computational determination of the solubility properties for thiostrepton
and its derivatives ............................................................................... 56
2.2.3.3. Inhibition of bacterial growth ............................................................. 56
2.2.3.4. In vitro inhibition of protein synthesis ............................................... 57
2.2.3.5. Reaction of thiostrepton with ionic copper ......................................... 57
2.3. Results and discussion .......................................................................................... 58
2.3.1. Synthesis of thiostrepton derivatives ............................................................ 57
2.3.2. Purification & characterization of thiostrepton derivatives .......................... 59
2.3.2.1. Optimization of purification conditions ............................................. 58
2.3.2.2. Physical characterization of the thiostrepton-MENSA derivative ...... 61
2.3.2.3. Modification of thiostrepton by bulky, polar thiols ............................ 65
2.3.2.4. Aqueous solubility of thiostrepoton derivatives ................................. 67
2.3.3. Biological activity of various thiostrepton derivatives ................................. 69
2.3.4. Thiostrepton-copper interactions .................................................................. 75
2.4. Conclusions, perspectives and future work .......................................................... 82

CHAPTER 3: DETERMINANTS OF ADOMET BINDING AND METHYL
GROUP TRANSFER FOR THIOSTREPTON RESISTANCE
METHYLTRANSFERASE
3.1. Preface .................................................................................................................. 84
3.2. Materials and methods ......................................................................................... 85
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3.2.1. Chemicals and reagents ................................................................................ 85
3.2.1.1. PCR and DNA manipulation .............................................................. 85
3.2.1.2. Media ................................................................................................. 86
3.2.1.3. Enzyme substrates .............................................................................. 86
3.2.2. Equipment .................................................................................................... 87
3.2.2.1. Incubators ........................................................................................... 87
3.2.2.2. Centrifugation..................................................................................... 87
3.2.2.3. Cell disruption .................................................................................... 87
3.2.2.4. Chromatography ................................................................................. 87
3.2.2.5. Spectrophotometry ............................................................................. 88
3.2.2.6. Fluorescence spectroscopy ................................................................. 88
3.2.2.7. Isothermal titration calorimetry .......................................................... 89
3.2.2.8. Mass spectrometry .............................................................................. 89
3.2.2.9. Polymerase chain reaction .................................................................. 89
3.2.3. General methods ........................................................................................... 89
3.2.3.1. Electrophoresis ................................................................................... 89
3.2.3.2. Protein mass spectrometry .................................................................. 90
3.2.3.3. Protein quantification ......................................................................... 91
3.2.3.4. DNA manipulation ............................................................................. 91
3.2.3.5. Site directed mutagenesis of TSR ....................................................... 91
3.2.3.6. Expression, purification and characterization of TSR mutants .......... 95
3.2.3.6.1. Size exclusion chromatography ................................................ 98
3.2.3.7. RNA methylation by TSR .................................................................. 98
3.2.3.8. Isothermal titration calorimetry .......................................................... 99
3.3. Results and discussion .......................................................................................... 101
3.3.1. Development of the coupled fluorometric assay .......................................... 101
3.3.2. Effect of point mutations on TSR activity .................................................... 103
3.3.3. Enzyme kinetics of AdoMet for TSR mutants ............................................. 108
3.3.4. AdoMet binding in point mutants of TSR .................................................... 113
3.4. Conclusions, perspectives and future work ........................................................... 120

CHAPTER 4: SYNTHESIS OF A NOVEL ADOMET ANALOGUE AND ITS
EVALUATION AS AN ADOMET METHYLTRANSFERASE CO-
SUBSTRATE
4.1. Preface .................................................................................................................. 123
4.2. Materials and methods .......................................................................................... 125
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4.2.1. Chemicals and reagents ................................................................................ 125
4.2.2. Equipment .................................................................................................... 125
4.2.2.1. Chromatography ................................................................................. 125
4.2.2.2. Spectrophotometry ............................................................................. 125
4.2.2.3. Mass spectrometry .............................................................................. 126
4.2.2.4. Gas chromatography .......................................................................... 126
4.2.2.5. Nuclear magnetic resonance ............................................................... 126
4.2.3. General methods ........................................................................................... 127
4.2.3.1. Synthesis of 3-iodo-1,1,1-trifluoroprop-2-one ................................... 127
4.2.3.2. Synthesis of S-adenosyl-S-(1,1,1-trifluoropropan-2-one)-L-methionine
(4.1) .................................................................................................... 127
4.2.3.3. Synthesis of the thiopurine methyltransferase product standard
(1,1,1trifluoro-3-[(4-nitrophenyl)thio]propane-2-one) (4.3) .............. 129
4.2.3.4. TMPT activity assay ........................................................................... 129
4.2.3.5. Competition assays ............................................................................. 131
4.3. Results and discussion .......................................................................................... 131
4.3.1. Synthesis of S-adenosyl-S-(1,1,1-trifluoropropan-2-one)-L-methionine (4.1)
................................................................................................................................ 131
4.3.2. Optimization of the reaction conditions for the synthesis of 4.1 .................. 135
4.3.2.1. General conditions .............................................................................. 135
4.3.2.2. Silver salts .......................................................................................... 136
4.3.2.3. Synthesis of 3-iodo-1,1,1-trifluoroprop-2-one (4.3) and its reaction with
AdoHcy .............................................................................................. 137
4.3.2.4. Attempted synthesis of activated trifluoromethyl ketones ................. 140
4.3.3. Evaluation of the trifluoropropane-2,2-diol AdoMet derivative (4.2) as a
potential Mtase co-substrate ......................................................................... 141
4.3.3.1. Thiopurine methyltransferase activity with 4.2 .................................. 141
4.4. Conclusions, perspectives and future work ........................................................... 149

CHAPTER 5: MOLECULAR INTERACTIONS BETWEEN THIOSTREPTON
AND THE TIPAS RESISTANCE PROTEIN FROM STREPTOMYCES
LIVIDANS
5.1. Preface .................................................................................................................. 154
5.2. Materials and methods .......................................................................................... 155
5.2.1. Chemicals and reagents ................................................................................ 155
5.2.2. Equipment .................................................................................................... 155
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