EVOLUTION AND PREVENTION OF ANTIBIOTIC RESISTANCE

172 Pages·2013·2.49 MB·English

by  Eliza Peterson

EVOLUTION AND PREVENTION OF ANTIBIOTIC RESISTANCE: SMALL
MOLECULE INHIBITORS OF BACTERIAL RECOMBINATION ENZYMES

Eliza J.R. Peterson

A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill
in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the
Department of Biochemistry and Biophysics

Chapel Hill
2013

Approved by:

Scott F. Singleton, Ph.D.

Ann H. Erickson, Ph.D.

Miriam Braunstein, Ph.D.

Charles W. Carter Jr, Ph.D

Edward J. Collins, Ph.D.
Abstract

ELIZA J.R. PETERSON: Evolution and Prevention of Antibiotic Resistance: Small
Molecule Inhibitors of Bacterial Recombination Enzymes
(Under the direction of Scott F. Singleton)

Antibiotic resistant bacteria are an ever-increasing problem for the modern
chemotherapy of bacterial infectious diseases. The loss of effective antibiotic therapies
due to antibiotic resistance and the withering antibiotic pipeline are resulting in a
reemergence in deaths from bacterial infections. New strategies are needed to combat
pathogenic bacteria and in this context bacterial targets involved in the development of
resistance are emerging an intriguing candidates for inhibition studies. Recent evidence
suggests that bacterial stress response pathways (i.e., SOS and competence for
transformation) are responsible for accelerated genetic changes that ultimately establish
antibiotic resistance. Intervening in these pathways by small molecule inhibition of key
recombination enzymes, RecA and EndA, would impact the DNA repair, SOS
mutagenesis and recombination-based horizontal gene transfer activities of these
enzymes and hinder the acquisition of antibiotic resistance. Bacteria having loss-of-
function mutations in the recA gene are more sensitive to antibiotic treatment and
develop resistance more slowly or not at all. In addition, endA-null strains of S.
pneumoniae have diminished transformation efficiencies and are unable to acquire
resistance-conferring DNA. Therefore, we believe chemotherapeutic agents that impart
these bacterial phenotypes could act synergistically with currently prescribed antibiotics
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to prevent the accumulation of populations that are resistant to them. Towards this goal,
we sought to identify properly designed inhibitors of RecA and EndA. High-throughput
screening (HTS) is recognized as a powerful tool in drug discovery to identify target-
specific lead compounds. We developed rational high-throughput screening programs to
discover small-molecule inhibitors of RecA and EndA. Through these studies, we have
identified novel chemical classes that specifically target RecA or EndA and demonstrate
that these enzymes hold potential as novel targets in the treatment of bacterial infections.

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Acknowledgements
I am grateful for the opportunity I have had to pursue my doctorate degree at the
University of North Carolina and want to thank those who been instrumental in my time
here. First, I thank my advisor, Dr. Scott Singleton, for his support and guidance
throughout my graduate career. Scott has taught me more than I realize and in particular
how to be a careful and critical scientist. His combination of intellect, professionalism,
and kindness is incredible. I especially appreciate his willingness to let me explore areas
of interest to me. I cannot remember a time he said “no” to any of my ideas, including
attending a conference in Brazil!
I extend thanks to the members of my thesis committee, Dr. Ann Erickson, Dr.
Miriam Braunstein, Dr. Charlie Carter and Dr. Ed Collins. Their thoughtful suggestions
and wise advice were instrumental to my progress in graduate school. More thanks go
out to important collaborators and scientific mentors at the Center for Integrative
Chemical Biology and Drug Discovery, Dr. Dmitri Kireev and Bill Janzen. Their
knowledge and expertise were an amazing resource during my training. I have enjoyed
the company of past and present members of the Singleton research group and several
deserve special mention for their help and friendship: Dr. Anna Gromova, Dr. Justin
Richards, Dr. John Bauman, Dr. Mike Jones, Dr. Lisa Heimbach, Morgan Chapman and
Demet Guntas.
I was very fortunate to make some great friends while in graduate school and wish
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them every success and happiness in the future. To Blaire Steinwand, thank you for
sharing this journey with me. It is difficult to imagine graduate school without the laughs,
tears, and bicycle rides we shared together.
Above all, I would like to thank my wonderful family for their love and support
throughout graduate school and my entire life. My parents, Jane and Paul, and my
sisters, Amy and Carrie, are all amazing people and remind me what is important in life.
I would finally like to acknowledge Nick Fey, Jeff Dunfee and my favorite little people,
Ana, Eloise, Oscar and Matilda Dunfee.

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Preface
Parts of this work were done in collaboration with other talented scientists.
Chapter 2 represents work done in collaboration with the Center for Integrative Chemical
Biology and Drug Discovery at the University of North Carolina. I am the lead author on
the paper and with the help of Dr. Scott Singleton, I designed and performed all
experiments and contributed to a majority of the writing. William Janzen provided
valuable guidance during RecA Transcreener assay optimization and Dr. Dmitri Kireev
assisted with analysis and clustering of active compounds. The paper was published
previous to writing this dissertation with the following citation:
Peterson EJ, Janzen WP, Kireev D, Singleton SF. High-throughput
screening for RecA inhibitors using a transcreener adenosine 5'-O-
diphosphate assay. Assay Drug Dev Technol. 2012. Jun;10(3):260-8).

Permission to include the article in its entirety in a PhD dissertation was retained from
Mary Ann Liebert, Inc (publisher of Assay Drug Dev Technol).
Chapter 3 represents an unpublished analysis of RecA HTS data performed
primarily by myself. Other members of the Singleton lab contributed to producing the
data used in the analysis. Morgan Chapman performed most of the SOS induction
experiments and Demet Guntas produced the majority of antibiotic potentiation data. Dr.
John Bauman, a former member of the lab, also assisted in producing some data as well.
Chapter 4 also presents unpublished data performed primarily by myself. Some
of the thermal denaturation studies were performed in the lab of Prof. Dev Arya at
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Clemson University. I received guidance from Dr. Nichola Garbett and Prof. Jonathan
Chaires at University of Louisville in developing the competition dialysis assays. I also
received valuable assistance from Victoria Madden, a research analyst at UNC’s
Microscopy Services Laboratory, with imaging using Transmission Electron Microscopy
Finally, Chapter 5 represents a publication for which I was sole first author. We
again collaborated with CICBDD and authors WPJ and DK provided the same assistance
as previous (Chapter 2). Dr. Marika Midon and Dr. Alfred Pingoud provided guidance
with the EndA plasmid conversion assay and characterizing the EndA variants. Andrea
Moon and Dr. Lars Pedersen purified the EndA (H160G) protein that was used in all
assays for EndA activity. With Dr. Singleton’s help, I designed the experiments,
executed them, and then wrote the paper. The paper was published previous to writing
this thesis with the following citation:
Peterson EJ, Kireev D, Moon AF, Midon M, Janzen WP, Pingoud A, Pedersen
LC, Singleton SF. Inhibitors of Streptococcus pneumoniae surface endonuclease
EndA discovered by high-throughput screening using a PicoGreen fluorescence
assay. J Biomol Screen. 2013 Mar;18(3):247-57.
Permission to include the article in its entirety in a PhD dissertation was retained from
Sage publications (publisher of JBS). Additional EndA screening of 960 compounds
from an EndA-focused set assembled by virtual screening methods was also performed
but not included in Chapter 5. These results are in preparation for submission to the
Journal of Medicinal Chemistry. Dr. Dmitri Kireev designed and performed all the
virtual screening approaches. My contributions to the work included all design and
execution of EndA activity assays. Dr. Dmitri Kireev, co-first author on the paper, has
given permission for me to include this work in my dissertation as Appendix I.

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Table of Contents
List of Figures .................................................................................................................... xi
List of Tables ................................................................................................................... xiv
List of Abbreviation .......................................................................................................... xv
Chapter 1 ............................................................................................................................. 1

1.1. Challenges of antibacterial discovery .................................................................................. 1

1.2. A revised model for accelerated evolution of antibiotic resistance...................................... 3

1.3. The role of recombination enzymes in accelerated evolution of antibiotic
resistance ..................................................................................................................................... 5

1.3.1. Induction of the SOS response and competence for transformation by antibiotics ...... 6

1.3.2. RecA and the SOS response ......................................................................................... 9

1.3.3. EndA and competence for transformation .................................................................. 10

1.4. Other roles of RecA and EndA in bacterial pathogenicity ................................................. 12

1.4.1. RecA ........................................................................................................................... 12

1.4.2. EndA ........................................................................................................................... 12

1.5. Intervening in accelerated evolution by small molecule inhibition of
recombination enzymes............................................................................................................. 14

1.6. Our strategy to develop small molecule inhibitors of recombination enzymes ................. 16
Chapter 2 ........................................................................................................................... 20

2.1. Introduction ........................................................................................................................ 20

2.2. Assay development and optimization ................................................................................ 23

2.3. Screen design and pilot study of LOPAC library ............................................................... 26

2.4. Screening 113,477 compounds for RecA inhibition .......................................................... 29

2.5. Follow-up evaluation of select inhibitors ........................................................................... 33
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2.6. Conclusions ........................................................................................................................ 34

2.7. Materials and Methods ....................................................................................................... 36
Chapter 3 ........................................................................................................................... 43

3.1. Introduction ........................................................................................................................ 43

3.2. Evaluation of compound attrition in hit generation stage .................................................. 44

3.3. Relationship between enzyme inhibition and in vitro bacterial activity ............................ 48

3.3.1 Antibiotic potentiation ................................................................................................. 48

3.3.2 Inhibition of SOS activation ........................................................................................ 52

3.4. Identification of prospective compounds for follow-up as “hits” for RecA
inhibition ................................................................................................................................... 56

3.5. Conclusion ......................................................................................................................... 58

3.6. Materials and Methods ....................................................................................................... 59
Chapter 4 ........................................................................................................................... 62

4.1. Introduction ........................................................................................................................ 62

4.2. Structural modifications aimed at selective inhibition of RecA ........................................ 63

4.3. Investigating DNA binding by styrylquinazolines and progenitor compounds
using thermal denaturation ........................................................................................................ 68

4.4. Evaluation of direct ligand DNA interactions using competition dialysis ......................... 72

4.5. Evaluation of small molecule colloidal aggregate formation ............................................. 76

4.6. Conclusions ........................................................................................................................ 81

4.7. Materials and methods ....................................................................................................... 82
Chapter 5 ........................................................................................................................... 86

5.1. Introduction ........................................................................................................................ 86

5.2. Assay development and validation..................................................................................... 89

5.3. Pilot screen of LOPAC library ........................................................................................... 97

5.4. Kinase focus set screening ............................................................................................... 100

5.5. Biochemical characterization of confirmed actives ......................................................... 104
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5.6. Conclusions ...................................................................................................................... 108

5.7. Materials and methods ..................................................................................................... 110
Chapter 6 ......................................................................................................................... 116
Appendix A ..................................................................................................................... 120

A.1. Introduction ..................................................................................................................... 120

A.2. Multiple virtual screening approaches ............................................................................ 122

A.3. Ad hoc substructure and pharmacophore search ............................................................. 124

A.4. Similarity search and Naïve Bayes learning ................................................................... 126

A.5. Structure-base screening ................................................................................................. 129

A.6. Hit analysis ...................................................................................................................... 130

A.7. Conclusions ..................................................................................................................... 134

A.8. Materials and methods .................................................................................................... 134
References ....................................................................................................................... 140

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