Pharmacognosic Drug Discovery with the Yersinia pestis MEP Synthase (IspC), a Validated Target for the Development of Novel Antibiotics
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Dowdy, Tyrone M
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Abstract
This thesis explores the role that a renewal in natural product research has in developing countermeasures to emergent antimicrobial resistance. Pharmacognosy, or the study of natural products, was the foundation of the antibiotic era in early 1900s. Due to convergent evolution and competition, organisms have become highly specialized in biosynthesizing secondary metabolites with specific targets in key metabolic pathways of pathogens, parasites, and competitors. Bioprospecting the metabolome of diverse samples of plants, fungi, and bacteria has provided a reliable resource for drug discovery and advancements in biomedical research. In this project, targeted high-throughput molecular screening has been used to bioprospect natural product extracts containing phytochemicals isolated from a variety of plants harvested from diverse ecosystems. Our aim was to identify novel inhibitors of the enzyme MEP synthase in the non-melavonate pathway for biothreat category A pathogens, such as Yersinia pestis. MEP synthase catalyzes the first committed step in the non-melavonate pathway and is the target enzyme in this investigation. The initial high-throughput screen resulted in four hits of interest, the most potent of which was natural extract 29 (e29). Ultracentrifugation affinity chromatography combined with quadrupole time-of-flight tandem mass spectrometry was used to identify the active component (quercetin) within e29. Subsequent enzyme assays confirmed the inhibitory activity of quercetin. Additionally, unlike other known inhibitors of MEP synthase, the mechanism of inhibition assays revealed that quercetin was an allosteric inhibitor of MEP synthase. Bacterial growth inhibition assays also demonstrated the effectiveness of quercetin as an antibiotic.
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MEP Synthase, Drug discovery, Yersinia pestis, Natural products, Pharmacognosy, Non-melavonate pathway