Structural features of fluoroquinolone-class antibiotics

206 Pages·2015·4.59 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2012
Structural features of fluoroquinolone-class
antibiotics that affect lethal activities and DNA
binding
Heidi Ann Schwanz
University of Iowa
Copyright 2012 Heidi Ann Schwanz
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/1395
Recommended Citation
Schwanz, Heidi Ann. "Structural features of fluoroquinolone-class antibiotics that affect lethal activities and DNA binding." PhD
(Doctor of Philosophy) thesis, University of Iowa, 2012.
https://doi.org/10.17077/etd.h0w5k1c5
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of thePharmacy and Pharmaceutical Sciences Commons
STRUCTURAL FEATURES OF FLUOROQUINOLONE-CLASS ANTIBIOTICS
THAT AFFECT LETHAL ACTIVITIES AND DNA BINDING
by
Heidi Ann Schwanz
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Pharmacy (Medicinal and Natural Products Chemistry)
in the Graduate College of
The University of Iowa
1
July 2012
Thesis Supervisor: Associate Professor Robert J. Kerns
1
ABSTRACT
Fluoroquinolones, broad-spectrum bactericidal antibiotics, exert their effects by
inhibiting type II topoisomerases through the formation of a fluoroquinolone-DNA-
topoisomerase ternary complex. Recently, newer, structurally unique fluoroquinolones
have been shown to kill bacteria by promoting chromosomal fragmentation in the
presence and absence of protein synthesis, thus allowing fluoroquinolones to potentially
be used in the treatment of microorganisms that go into a dormant state. There is a need
to further understand the structure activity relationships (SAR) of fluoroquinolones to
develop new antibiotics that can kill dormant bacteria and are active against current
resistant strains. The hypothesis that structurally unique fluoroquinolones interact with
the DNA- fluoroquinolone-topoisomerase ternary complex in a unique way that leads to
different killing pathways is the basis of this work.
The first approach to understand SAR for fluoroquinolones to kill non-growing
bacteria was to evaluate the effect of modifications at the C-8 and C-5 positions on
lethality. Novel, synthetically-derived and commercially-available fluoroquinolones
were evaluated for ability to kill Escherichia coli in the presence and absence of
chloramphenicol, a known protein synthesis inhibitor used to simulate non-growing
bacteria.
The second study was to understand SAR of fluoroquinolone-class agents
1

necessary to maintain antibacterial activity against common fluoroquinolone resistance-
causing bacterial mutations on topoisomerase IV. A panel of novel fluoroquinolones,
2,4-quinazoline diones, and fluoroquinolone-like analogues with unique substitution
combinations at C-8 and C-7 was synthesized and evaluated for ability to poison wild-
type and mutant Bacillus anthracis topoisomerase IV.
The third study to understand the contribution of SAR of fluoroquinolone-class
agents to novel killing mechanisms was to evaluate the binding interaction of
2
fluoroquinolones to double-stranded and nicked DNA. Binding affinities of
fluoroquinolones to DNA were determined; fluoroquinolones were found to bind
different DNA types with varied affinities. The ability of a series of C-8 and C-7
modified fluoroquinolones to stabilize or destabilize DNA was assessed.
The results of these studies also add broadly to the understanding of SAR
associated with fluoroquinolone-class antibiotics for killing in the presence and absence
of protein synthesis, maintaining activity in the presence of resistance-causing mutations
in the target enzymes, and increasing binding interactions with different types of DNA.

Abstract Approved: ____________________________________
Thesis Supervisor
____________________________________
Title and Department
____________________________________
Date
2
STRUCTURAL FEATURES OF FLUOROQUINOLONE-CLASS ANTIBIOTICS
THAT AFFECT LETHAL ACTIVITIES AND DNA BINDING
by
Heidi Ann Schwanz
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Pharmacy (Medicinal and Natural Products Chemistry)
in the Graduate College of
The University of Iowa
1
July 2012
Thesis Supervisor: Associate Professor Robert J. Kerns
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Heidi Ann Schwanz
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Pharmacy (Medicinal and Natural Products Chemistry) at the July
2012 graduation.
Thesis Committee: ___________________________________
Robert J. Kerns, Thesis Supervisor
___________________________________
Jonathan A. Doorn
___________________________________
Zhendong Jin
___________________________________
David L. Roman
___________________________________
Marc S. Wold
To my mom for her guidance
To my family for their support
To both for their unconditional love
2

ii
ACKNOWLEDGMENTS
My most sincere gratitude goes to my advisor Dr. Robert Kerns for accepting me
into his laboratory and providing me with numerous opportunities to grow as a scientist.
I am grateful for his instruction and guidance throughout the course of my graduate
career. I would also like to thank the members of my committee - Dr. Jonathan Doorn,
Dr. Zhendong Jin, Dr. David Roman, and Dr. Marc Wold - as well as that rest of the
MNPC faculty and staff - Dr. Michael Duffel, Dr. Horacio Olivo, Dr. Kevin Rice, Kelly
Walsh, and Kellie Northup - for the guidance and assistance provided. Special thanks to
Dr. Duffel and Dr. Doorn for their letters of support for awards and my next career step.
I would like to acknowledge our collaborators, whose contributions are seen in
many of the figures and tables: Karl Drlica, Muhammad Malik, Hiroshi Hiasa, and Neil
Osheroff. I would also like to acknowledge the National Institutes of Health, Center for
Biocatalysis and Bioprocessing at the University of Iowa, and the American Foundation
for Pharmaceutical Education for providing me with prestigious fellowships throughout
my graduate career.
A big thank you to all the members of the Kerns laboratory over the years, as well
as the other graduate students in the division, for friendship and support that definitely
helped pull me through the highs and lows of graduate school. All of the advice and
encouragement when research was not going very well, as well as constructive
3

discussions and critiques, were much appreciated.
Finally, I would like to extend my thanks to the best family and friends I could
ask for. Thank you all for the tireless support and encouragement over the last five years.
Most importantly, thank you to my mom Roxie for being an amazing role model,
teaching me not to give up when times are tough, and showing me that I can do anything
I put my mind to. I love you all.
.
iii
ABSTRACT
Fluoroquinolones, broad-spectrum bactericidal antibiotics, exert their effects by
inhibiting type II topoisomerases through the formation of a fluoroquinolone-DNA-
topoisomerase ternary complex. Recently, newer, structurally unique fluoroquinolones
have been shown to kill bacteria by promoting chromosomal fragmentation in the
presence and absence of protein synthesis, thus allowing fluoroquinolones to potentially
be used in the treatment of microorganisms that go into a dormant state. There is a need
to further understand the structure activity relationships (SAR) of fluoroquinolones to
develop new antibiotics that can kill dormant bacteria and are active against current
resistant strains. The hypothesis that structurally unique fluoroquinolones interact with
the DNA- fluoroquinolone-topoisomerase ternary complex in a unique way that leads to
different killing pathways is the basis of this work.
The first approach to understand SAR for fluoroquinolones to kill non-growing
bacteria was to evaluate the effect of modifications at the C-8 and C-5 positions on
lethality. Novel, synthetically-derived and commercially-available fluoroquinolones
were evaluated for ability to kill Escherichia coli in the presence and absence of
chloramphenicol, a known protein synthesis inhibitor used to simulate non-growing
bacteria.
The second study was to understand SAR of fluoroquinolone-class agents
4

necessary to maintain antibacterial activity against common fluoroquinolone resistance-
causing bacterial mutations on topoisomerase IV. A panel of novel fluoroquinolones,
2,4-quinazoline diones, and fluoroquinolone-like analogues with unique substitution
combinations at C-8 and C-7 was synthesized and evaluated for ability to poison wild-
type and mutant Bacillus anthracis topoisomerase IV.
The third study to understand the contribution of SAR of fluoroquinolone-class
agents to novel killing mechanisms was to evaluate the binding interaction of
iv
fluoroquinolones to double-stranded and nicked DNA. Binding affinities of
fluoroquinolones to DNA were determined; fluoroquinolones were found to bind
different DNA types with varied affinities. The ability of a series of C-8 and C-7
modified fluoroquinolones to stabilize or destabilize DNA was assessed.
The results of these studies also add broadly to the understanding of SAR
associated with fluoroquinolone-class antibiotics for killing in the presence and absence
of protein synthesis, maintaining activity in the presence of resistance-causing mutations
in the target enzymes, and increasing binding interactions with different types of DNA.
5

v