Publication Date

2016

Document Type

Dissertation/Thesis

First Advisor

Horn, James R.||Hagen, Timothy J.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry

LCSH

Enzyme inhibitors; Anti-infective agents; Drug resistance in microorganisms

Abstract

There is an immediate need for new antibiotics. Antimicrobial resistance is rising at an alarming rate. In addition, certain select agents are a risk for bioterrorism that necessitate the discovery of new antibiotics. The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoid precursors, isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Notably, the MEP pathway is present in most bacteria and not mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to reduced chances of off-target interactions leading to side effects. Currently, there are very few known inhibitors of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) enzymes, with the majority possessing weak potency (IC₅₀ values in the millimolar range). The focus of this research examines the biophysical properties of IspD and IspE to aid discovery of novel inhibitors. Saturation transfer difference nuclear magnetic resonance (STD- NMR) fragment screening identified several fragments that bound to both IspD from Mycobacterium paratuberculosis and IspE from Mycobacterium abscessus. Analogs of these fragments aimed to target IspD and IspE. Thermal shift screening was used as a filter to identify a subset of compounds that bind to the IspD and IspE enzymes. High throughput thermal shift screening of the MicroSource Spectrum library against EcIspD revealed several potential hits, which were subsequently investigated using a plate?based enzyme inhibition assay. Results from the enzyme inhibition assays revealed a small set of compounds capable of inhibiting IspD activity. Follow-up studies were performed using isothermal titration calorimetry and enzymatic inhibition assays. Results from these studies have confirmed compounds with high micromolar inhibition targeting IspD from Escherichia coli. The IspE enzyme from both Escherichia coli and Burkholderia thailandensis were characterized with biophysical methods. Additionally, a plate-based enzyme inhibition assay was optimized to evaluate potential IspE inhibitors, which helped discover several novel inhibitors (IC₅₀ values in the micromolar range). The identified compounds could be used for future development of more potent IspD and IspE inhibitors.

Comments

Advisors: James R. Horn; Timothy J. Hagen.||Committee members: Gary M. Baker; Elizabeth R. Gaillard; Joel P. Stafstrom.||Includes bibliographical references.||Includes illustrations.

Extent

xviii, 150 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

Download

Share

COinS