Proteomic Approaches For Profiling Cofactor-Dependent Proteins In Mycobacterium Tuberculosis
2022-12
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Proteomic Approaches For Profiling Cofactor-Dependent Proteins In Mycobacterium Tuberculosis
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2022-12
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Tuberculosis is an infectious disease that currently affects a quarter of the world’s population. The rise of new antibacterial resistant strains makes the discovery and development of new antibiotics imperative. Chapter 1 provides a broad overview of PLP-dependent enzymes in Mycobacterium tuberculosis, the causative pathogen of tuberculosis. PLP-dependent enzymes represent an important class of highly “druggable” enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes have been validated as therapeutic targets for pathologies ranging from African sleeping sickness to epilepsy. A full catalog of all PLP-dependent enzymes in Mtb has been compiled and references provided on any drug discovery done at that target. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. In Chapter 2, we developed a probe that contains an N-hydroxy alanine warhead, which acts as a mechanism-based inhibitor of PLP-dependent enzymes, a linear diazirine for UV-crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was utilized to study PLP-dependent enzymes in vitro as well as look at whole cell lysates of Mycobacterium tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, via mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes to enable the characterization of rationally designed mechanism-based inhibitors of PLP-dependent enzymes which will expedite the preclinical characterization of these important therapeutic entities. Another area of interest for this work was that of nucleotide binding proteins. Nucleotides such as ATP, NAD, CoA, and SAM are critical in numerous enzymatic processes for eukaryotic and prokaryotic cells to sustain life In Chapter 3, we explore taking classical affinity-based chromatography techniques by way of an ATP-resin for the isolation of nucleotide binding proteins in Mycobacterium tuberculosis and pairing it with a modern proteomics protein identification strategy. We used a previously reported ATP-Sepharose resin provided by a collaborator to identify a vast number of proteins within the Mtb “NTPome” and observe improved coverage compared to previous approaches. We then were able to utilize the resin to profile selectivity of various adenylating enzyme inhibitors developed in our lab. Lastly, we sought to create a magnetic ATP-bead for a faster and more high-throughput workflow of this technique.
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University of Minnesota Ph.D. dissertation. December 2022. Major: Medicinal Chemistry. Advisor: Courtney Aldrich. 1 computer file (PDF); xxvii, 274 pages.
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Brody, Scott. (2022). Proteomic Approaches For Profiling Cofactor-Dependent Proteins In Mycobacterium Tuberculosis. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/260687.
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