Hagen, Timothy J.
Horn, James R.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
With the likelihood of contracting an antibiotic resistant infection increasing, the need for novel anti-infective agents is critical. The development of novel anti-infective agents benefits from the identification of new target pathways necessary for the survival of the pathogen. One such target is the methylerythritol phosphate (MEP) pathway, responsible for the synthesis of the isoprenoid precursors isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) in bacteria and other pathogenic organisms. The MEP pathway is an attractive target because it is specific to a wide range of pathogenic organisms, while humans have a separate pathway, the mevalonate (MVA) pathway, for the biosynthesis of IPP and DMAPP. The focus of the project is the characterization of potential inhibitors of the MEP pathway by targeting the fifth enzyme in the pathway, 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). In this study, the first goal was to develop and optimize high-throughput assays that can be used to evaluate the potency of compounds that target IspF. A novel IspF assay framework was devised using a commercially available assay kit that is currently under development. In addition, a previously reported assay was optimized for more efficient high-throughput screening (HTS). The novel framework utilized a single coupled enzyme step with malachite green (MG) as the observable read-out. While this assay possessed advantages, including the amount of each reagent used and the time necessary to perform the assay, the observable range was not sufficient to distinguish typical inhibition constants. The optimization of the previously reported assay came with its own set of challenges as the previously utilized kit was discontinued, so a similar kit was needed for assay development. Additional optimization is still needed for either to be a viable HTS options. Due to the complexities of the inhibition assays, a novel fluorescence-based competitive binding assay was developed. Having previous knowledge of ligand-IspF structures, synthesized fluorescent ligands were characterized by their ability to serve as competitive fluorescent probes in a competitive binding assay. The initial fluorescent ligand was found to have an apparent binding affinity (Kd,app) of about 100 µM by fluorescence anisotropy (FA). Simulations revealed this binding affinity was too weak to act as an effective probe. Consequently, additional modifications of the fluorescent ligand were explored. Two dansyl compounds were found to bind to Burkholderia pseudomallei IspF with Kd,app ≥ 10 µM. These ligands were compared against a derived competitive binding model to determine their applicability as competitive binding ligands and successfully acted as a probe in said assay against a test ligand. A previously identified IspF ligand, L-tryptophan hydroxamate, was investigated to determine the mode of binding and binding thermodynamics. Initial reports found that it bound to Escherichia coli IspF with a Kd,app of 2 µM by surface plasmon resonance (SPR). Investigation by isothermal titration calorimetry (ITC) revealed that L-tryptophan hydroxamate bound to B. pseudomallei and E. coli IspF with Kd,apps near 30 µM with some evidence of pH dependent binding. Structural analysis by x-ray crystallography revealed a non-traditional binding mode to the active site zinc. Traditionally, hydroxamic acids bind zinc in a trigonal bipyramidal geometry through the hydroxamate oxygens, but L-tryptophan hydroxamate binds in an apparent trigonal bipyramidal geometry through the amide and hydroxamate nitrogens. Investigations of other zinc binding moieties demonstrated that hydroxamic acids have the best zinc binding potential and other amino acid hydroxamates could bind to IspF. To identify new lead compounds targeting IspF, a high-throughput screening of the Spectrum Collection was conducted using a fluorescent thermal shift (FTS) assay. Of the 2,560 bioactive compounds and natural products, seventy had thermal shifts (ΔTm) ≥ 4°C. After filtering and removing pan-assay interference compounds (PAINS), sixteen compounds were chosen for further investigation. After examining these ‘hits’ through docking studies and saturation transfer difference nuclear magnetic resonance (STD-NMR) experiments, seven displayed interactions with B. pseudomallei IspF. Further analysis by ITC found that four sulfonamides, three of which were ‘hits’ in the Spectrum Collection screen, bound to IspF. Structural analysis also resulted in the four sulfonamides having co-crystal structures with B. pseudomallei IspF. Sulfonamides are known zinc binders and are reported inhibitors of carbonic anhydrase, a zinc metalloenzyme. Based on the screening of the Spectrum Collection, four sulfonamides were found to bind IspF and two were chosen for structure-activity relationship (SAR) development. A series of both sulfonamide analogs were synthesized and evaluated to determine functional groups that may enhance the ligand binding affinity to B. pseudomallei IspF. Examining each series resulted in five compounds with thermal shifts ≥ 4°C. Of these FTS ‘hits’, three showed binding to B. pseudomallei IspF by ITC and one ultimately resulted in a co-crystal structure through structural analysis. In conclusion, two novel assays were designed, developed, and preliminarily optimized for their use as IspF ligand screens. A previously reported ligand was investigated through structural and thermodynamic analysis to determine its binding mode and affinity towards IspF. A compound library was screened revealing four previously unreported ligands effectively bind IspF. Investigation of sulfonamide analogs and SAR development found a novel acetazolamide analog bound and co-crystalized with IspF. Overall, the binding modes of six ligands with B. pseudomallei IspF have been revealed, L-tryptophan hydroxamate and five sulfonamides. This knowledge helped in the development of a sulfonamide fluorescent ligand for a competitive binding assay. While further work is needed to optimize these assays and ligands, they provide a great framework for the design of future IspF inhibitors and fluorescent reagents.
Grote, Dakota Lane, "Characterization of Potential Anti-infective Agents By Targeting Burkholderia pseudomallei and Escherichia coli IspF" (2021). Graduate Research Theses & Dissertations. 7081.
Northern Illinois University
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