Hagen, Timothy J.
M.S. (Master of Science)
Department of Chemistry and Biochemistry
There is an urgent need to discover new antibiotics to combat the emergence of drug resistant Novel antibiotics that target unique pathways different from existing therapies are promising targets. The non-mevalonate pathway (MEP), which was discovered in the 1990's, is essential for most bacteria, plants and apicomplexan parasites. Inhibition of the non-mevalonate is an attractive pharmaceutical target, since it is not present in mammals. Many compounds have surfaced as potential inhibitors, but none have made it to market. This thesis investigates the fourth step in the MEP pathway involving the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from Escherichia coli (Ec), Burkholderia thailandensis (Bt), and Mycobacterium abscessus (Ma). The initial aim of this study was to identify in vitro conditions that stabilize the IspE enzyme and to identify interactions within IspE's CDP-ME and ATP binding sites that contribute significantly to the binding energetics. The conditions that best stabilized the enzymes were investigated with temperature melts using differential scanning fluorimetry and circular dichroism. All three IspE species displayed melting temperatures (Tm ) that heavily depended on pH, with Tm values dropping significantly with pH values less than 5.0. All three species preferred a reduced environment with high salt content, along with the presence of the metal co-factor Mg²⁺. These conditions were used to evaluate the binding thermodynamics of the substrate/co-factor derivatives with IspE using isothermal titration calorimetry. Results showed that the loss of phosphate groups on ATP most significantly impacted the binding constant. This suggests that the phosphate binding loop plays an important role in ligand binding. Targeting this region with small molecule compounds may help improve IspE inhibitor potency. Also, a significant thermal shift of 10°C with bound CMP to MaIspE suggests hydrogen bonding to SER147 contributes significantly to substrate binding. Isotopically-labeled CDP-ME was also synthesized enzymatically for future characterization of the IspE enzyme by NMR. It can be utilized to monitor conversion of substrate to product and for assay development. Finally, 4-quinazolinone compounds were evaluated as potential inhibitors of IspE activity. However, 4-quinazolinone compounds have been hypothesized to inhibit either IspE or the auxiliary enzyme luciferase, which is a downstream coupled enzyme in certain IspE assays. This study found the compounds did not inhibit the luciferase and could in fact be potent IspE inhibitors. The IspE inhibition assay did not reproduce all IC₅₀ values of various reference compounds which indicate that further investigation is necessary.
Hoerchler, Kataryzna Barbara, "Evaluation of IspE for inhibitor design in the non-mevalonate pathway" (2018). Graduate Research Theses & Dissertations. 2935.
Northern Illinois University
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