Date of Award

8-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

Hevener, Kirk

Committee

Sarka Beranova-Giorgianni, Isaac O. Donkor, Eric J. Enemark, C. Ryan Yates

Keywords

Antibacterial, Clostridioides difficile, Enoyl-ACP reductase, FabK, Narrow-spectrum, Topoisomerase I

Abstract

Background: Significant concerns are associated with the use of broad-spectrum antibacterial agents, including collateral eradication of beneficial bacteria from the human microbiome, the onset of antibacterial-associated infections, and continued emergence of antibacterial drug resistance. As such, a critical need for novel and selective antibacterial targets exists. The investigation of two such targets, each pertaining to the highly concerning infections caused by streptococcal species and Clostridioides difficile, are presented herein. Bacterial topoisomerase I represents a potentially promising narrow-spectrum target as studies have arisen demonstrating its essentiality in bacterial species lacking the only other type IA topoisomerase (topoisomerase III). Additionally, recent studies demonstrating the essentiality of the fabK gene expressing enoyl-ACP reductase II (FabK) in C. difficile indicate its significant potential as a narrow-spectrum target. Presented here are data characterizing and validating both the TopoI and FabK enzymes as novel antibacterial targets via the implementation of an array of drug discovery techniques, including structural studies, biochemical assay development and application, and inhibitor screening and testing. Methods: An assortment of drug discovery techniques were employed for the targeting of SmTopoI and CdFabK, including different protein expression and purification techniques; X-ray crystallography; various biophysical and biochemical techniques for target characterization, validation, and drug screening; and different lead development and optimization studies. Results: The respective genes for SmTopoI and CdFabK have been cloned, and the expression and purification of various constructs of each target have been carried out and optimized for further analysis. The crystal structure of SmTopoI_N65 has been determined to 2.06 Å and diffracting CdFabK crystals (3.5 Å) have been attained. A high-throughput plate-based biochemical fluorescence kinetic assay has been optimized for screening against the CdFabK enzyme. Furthermore, activity and modality of inhibition assessment of small-molecule inhibitors of the CdFabK enzyme have been conducted, including phenylimidazole and benzothiazole compounds. Phenylimidazole analogues have been found to display micromolar inhibitory activity against CdFabK, and a benzothiazole analogue has been found to display nanomolar inhibitory activity against the target. Conclusions: The SmTopoI and CdFabK enzymes present potentially novel, narrow- spectrum antibacterial drug targets, and substantial progress has been made toward the rational targeting of these two enzymes. Of particular note, the first structure of a Topo I fragment from a gram-positive organism, S. mutans, has been determined. Enzymology and inhibitor studies have been conducted supporting the druggability of CdFabK and indicating the potential for selective inhibition of CdFabK.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0001-8006-5661

DOI

10.21007/etd.cghs.2019.0491

Additional Files
2019-014-Ross-DOA.pdf (441 kB)
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