Document Type


Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Robert J. Kerns


Fluoroquinolones, a class of type-II topoisomerase inhibitors, have successfully been used as antibiotics for the last several decades, beginning with the use of nalidixic acid in urinary tract infections. This led to the broad-spectrum activity of ciprofloxacin in the 1980s. Unfortunately, use of fluoroquinolones has led to the emergence of resistant bacteria. Recently, this has generated new bacteria such as multidrug-resistant and extensive-drug-resistant strains of M. tuberculosis that are also fluoroquinolone-resistant. Infections caused by these bacterial strains are widespread, with high mortality rate in immune-compromised populations such as the elderly, infants, and in AIDS or HIV-positive patients.

Fluoroquinolone resistance is acquired through amino acid substitutions of key fluoroquinolone-binding residues of the type-II bacterial topoisomerases DNA Gyrase and Topoisomerase IV, the enzyme targets of fluoroquinolones. Amino acid substitutions that result in fluoroquinolone resistance are located on Helix-4 of these enzymes, which is the site of a magnesium (Mg)-water bridge that is a crucial binding interaction for fluoroquinolones. When certain substitutions to Helix-4 occur, the Mg-water bridge is compromised and no longer available to anchor fluoroquinolones into a ternary complex composed of topoisomerase, fluoroquinolone, and DNA. This results in drug resistance. Herein are described attempts to generate fluoroquinolones that are capable of overcoming this mechanism of resistance.

In the first study, attempts were made to generate a series of novel tricyclic fluoroquinolones and diones designed to exploit intercalative or pi-stacking binding interactions with the bacterial DNA in the ternary complex in order to lessen the importance of the Mg-water bridge interaction. Despite numerous attempts, no complete synthetic pathway to these core structures was ever discovered.

The second study investigated the utility of a C7-aminomethylpyrrolidine group on the fluoroquinolone structure. This was done in order to explore the mechanistic reasons why previously generated fluoroquinolones possessing this C7-aminomethylpyrrolidine group maintained activity against common Helix-4 mutants. A panel of fluoroquinolones with C7-aminomethylpyrrolidine groups and diverse core structures was synthesized and docking studies with the original C7-aminomethylpyrrolidine fluoroquinolone and other fluoroquinolones were performed. Target compounds were synthesized and evaluated for inhibition/poisoning purified enzyme and for the ability to inhibit growth with wild-type and fluoroquinolone-resistant cells. In a third study, fluoroquinolones possessing structural variations of the C7-aminomethylpyrrolidine were designed and synthesized to explore structural requirements of the aminomethylpyrrolidine group binding and overcoming fluoroquinolone-resistance caused by alterations of Helix-4. This led to further exploration of the binding space around the C7-position of the fluoroquinolones. In both the second and third studies, the new fluoroquinolones were evaluated for the ability to specifically target bacterial topoisomerases over human topoisomerase. The results of these studies have contributed new knowledge to the binding requirements of fluoroquinolones that maintain potency against fluoroquinolone-resistant type-II topoisomerases, and represent a step towards methodology to overcome bacteria resistant to fluoroquinolones.

Public Abstract

Fluoroquinolones are highly successful antibiotics that, through widespread use, suffer diminishing clinical utility due to emerging antibiotic resistance. The emergence of fluoroquinolone-resistant bacteria is believed to be caused by evolution-driven changes in the targets of fluoroquinolone action, namely the type-II bacterial topoisomerases DNA Gyrase and Topoisomerase IV. The function of type-II topoisomerases is the untangling of knots in bacterial DNA and the separation of newly replicated bacterial DNA. Type-II topoisomerases are crucial to the replication of bacterial DNA, and ultimately cellular reproduction in bacteria. Fluoroquinolones are able to bind into a structure composed of bacterial DNA bound into topoisomerase. This entire structure, which is composed of fluoroquinolone, DNA, and topoisomerase, is called the ternary complex. The binding of the fluoroquinolone into the ternary complex stops the process of DNA untangling by topoisomerase, which in turn halts cellular growth and leads to cell death. Fluoroquinolones that are able to stabilize the ternary complex are referred to as topoisomerase poisons.

The goal of the research described herein is the development of new fluoroquinolones that are able to poison both wild-type bacteria and fluoroquinolone-resistant bacteria. This was accomplished by the generation of fluoroquinolones that possess side chain structures that bind to places in the ternary complex that are separate from the resistance-causing alterations within topoisomerase. The ability of these new fluoroquinolones to 1) poison bacterial topoisomerases and 2) not poison human topoisomerase was tested in purified bacterial and human topoisomerase enzymes. The ability of these new fluoroquinolones to inhibit bacteria cell growth was tested in wild-type and fluoroquinolone-resistant cell cultures.


publicabstract, Aminomethylpyrrolidine, Fluoroquinolone, Topoisomerase


xix, 153 pages


Includes bibliographical references (pages 146-153).


Copyright 2015 Benjamin Howard Williamson