Structure, mechanism, and inhibition of proline metabolic enzymes
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Proline metabolic enzymes have been the focus of inhibitor discovery due to their role in cancer cell proliferation and metastasis. The proline biosynthetic enzyme PYCR1 has been associated with high tumor expression, which is linked to poor patient outcomes and tumor aggressiveness. The main focus of this thesis is discovery of novel compounds to inhibit proline biosynthetic and proline catabolic enzymes through structure-based and fragment-based inhibitor discovery methods. These compounds can be used as chemical probes to further elucidate the role of proline metabolism in cancer. Chapter 2 focuses on the optimization of expression and purification of PYCR3, which had previously been insoluble or difficult to purify. Extensive kinetics were performed to determine the mechanism of PYCR3 is most likely the random bi bi mechanism. Additionally, a library of proline analogs was screened against PYCR3. A known PYCR1 inhibitor was found to be 10 times more selective for PYCR1 over PYCR3. A crystallography-based fragment screen performed on PRODH and GSALDH is the focus of Chapter 3. A 288-fragment library was screened in crystallo in cocktails of six fragments. A novel PRODH inhibitor, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site. Analog screening of 4-methoxybenzyl alcohol led to the discovery of a more potent inhibitor of PRODH than the initial hit fragment. Chapter 4 details the screening of 71 fragments against PYCR1 using an enzyme activity assay. Twelve hit compounds were validated with X-ray crystallography. The library was counterscreened against PYCR3 and PRODH. (S)-tetrahydro-2H-pyran-2-carboxylic acid has higher affinity for PYCR1 than NFLP, is 30 times more specific for PYCR1 than PYCR3, and negligibly inhibits PRODH. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate provide the first evidence that the sulfonate group is a suitable replacement for the carboxylate anchor for these enzymes. Chapter 5 serves as a proof-of-concept study for template-based docking on our PYCR1 system. 37 fragment-like carboxylic acids were screened using X-ray crystallography. Strong electron density was observed for 8 compounds, corresponding to a 22% hit rate. The fragments are the first PYCR1 inhibitors to block the P5C and NAD(P)H site simultaneously. This chapter also highlights the importance of crystallography as the primary screening method as only 4 of the 8 hit compounds showed inhibition in enzyme activity assays, highlighting the distinction between binders and inhibitors. 5-oxo-7a-phenyl-hexahydropyrrolo[2,1-b][1,3]thiazole-3-carboxylic acid had a lower IC50 than our benchmark compound NFLP. Combining the success of template-based docking in Chapter 5 and replacement of the carboxylate anchor in Chapter 4, Chapter 6 details a carboxylate bioisosteric library of 22 compounds. Four compounds were found to bind PYCR1 in novel remote binding sites, and inhibit PYCR1 activity, classifying them as allosteric inhibitors. Finally, Chapter 7 contains preliminary data of drug-like molecules screened in a more high throughput approach with enzyme activity assays as the primary screen. 24 compounds appear to inhibit PYCR1 more than NFLP. However, these compounds should be further validated with X-ray crystallography in future studies.
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Ph. D.