Publication Date

2016

Document Type

Dissertation/Thesis

First Advisor

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

Degree Name

Ph.D. (Doctor of Philosophy)

Department

Department of Chemistry and Biochemistry

LCSH

Anti-infective agents||Antibiotics--Synthesis||Drug resistance in microorganisms||Drug resistance in microorganisms--Immunology

Abstract

There is an urgent need for new anti-infective agents. Pathogenic organisms continue to develop resistance to modern-day antibiotics, while there has been a significant decline in the development of antibiotics over the last 30 years. One particular pathway of interest in the development of new antibiotics is the biosynthesis of isoprenoids. These compounds are essential chemical building blocks in pathogenic organisms such as Plasmodium falciparum, Mycobacterium tuberculosis, and Burkholderia pseudomallei. Notably, there are two distinct pathways in the biosynthesis of isoprenoids called the Mevalonate (MVA) pathway and the 2C-methyl-D-erythritol phosphate (MEP) pathway. The MEP pathway enzymes are attractive targets in developing novel anti-infective agents due to the fact that humans only use the MVA pathway to produce isoprenoids. Burkholderia pseudomallei is a Gram-negative bacterium found in untreated water and soil in Southeast Asia and northern Australia. The bacterium has been classified as a category B bioterrorism agent/disease by the Centers for Disease Control and Prevention. Antibiotic resistance to traditional anti-microbial agents in such pathogenic organisms has increased due to the primary mechanisms of resistance, which are enzyme inactivation, drug efflux from the cell, and target deletion. Consequently, new anti-infective agents targeting B. pseudomallei are of great interest. The focus of this research project was to examine the inhibition of the fifth enzyme in the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) using small molecules that were developed based on fragment hits and structure activity relationships (SAR). In this study, enzyme engineering was utilized for the development of a high throughput thermal-shift assay. The assay was used to assess the relative potency of compounds targeting IspF. The inhibitor binding affinities and thermodynamics were determined using Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). Enzyme inhibition values for the most potent inhibitors were then evaluated using a High Performance Liquid Chromatography (HPLC) activity assay. Finally, structural analysis for two compounds in complex with IspF was completed to assess the mode of binding in the active site, which will aid in future inhibitor design strategies.

Comments

Advisors: James R. Horn; Timothy J. Hagen.||Committee members: Gary M. Baker; Oliver Hofstetter; Richard L. Walter.||Includes bibliographical references.||Includes illustrations.

Extent

xxi, 226 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

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