Hagen, Timothy J.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
This body of work focuses on the inhibition of Burkholderia pseudomallei enzyme IspF. Burkholderia pseudomallei is the causative agent of Whitmore's disease, and IspF is an essential enzyme in the synthesis of isoprenoids, a vital class of compounds. This work has involved the design and synthesis of nearly five dozen compounds, as well as the design, modification, and use of assays to demonstrate potential interactions between the compounds being investigated and the targeted organisms or enzymes. Development of assays was made challenging by the difficulty in observing the consumption of an unstable substrate or production of a product without a chromophore. Five series of compounds were synthesized. First, a series of pyrimidine derivatives with a zinc binding scaffold was synthesized and conclusively shown to have antibacterial activity against both Gram-positive and Gram-negative bacteria. Several of these compounds were shown to interact with Burkholderia pseudomallei IspF mutant Q151E via a fluorescence-based thermal shift assay. Furthermore, group epitope mapping by nuclear magnetic resonance (NMR) was used to determine the binding characteristics of compound 38 in this series. A second series of compounds was designed as derivatives of a weakly binding ligand containing dansyl, imidazole, and phenol moieties. These derivatives maintained the fluorescent dansyl moiety and their dissociation constants were determined by fluorescence anisotropy. This led to the discovery of the first submicromolar BpIspF ligand. This compound could potentially be used as a probe in future displacement fluorescence anisotropy assays to determine the dissociation constants of non-fluorescent compounds. A series of compounds originally identified through a library screening were investigated by NMR to validate the screening results. Saturation transfer difference NMR (STD NMR) experiments revealed that almost all of the hits or a degradation product formed in solution interact with the protein. Interactions of compounds with the protein were mapped, showing the regions of the compound most important to binding. A series of compounds based on one of the hits, ethoxzolamide, was also synthesized. Preliminary data on several of these compounds have displayed antibacterial activity and Salmonella typhimurium IspF enzyme inhibition greater than that of the parent compound. Derivatives of known IspF ligands were also synthesized with intent to improve upon their characteristics and activity. These compounds displayed antibacterial activity against nine organisms, Gram-negative and Gram-positive bacteria alike. Additional ligands were also explored through group epitope mapping to support or reveal previously unknown binding characteristics. The output of this work can be used for future projects to develop more potent compounds in the pursuit of Burkholderia pseudomallei IspF inhibitors, as well as inhibitors of other organisms.
Watkins, Sydney, "Design, synthesis, and evaluation of potential Burkholderia pseudomallei IspF inhibitors" (2019). Graduate Research Theses & Dissertations. 2119.
Northern Illinois University
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