Trichomonas vaginalis is an obligate protozoan parasite that infects over 400 million people worldwide, with an increasing number of infections resistant to current treatments. Since the parasite cannot synthesize its own nucleobases, nucleoside salvage pathway enzymes represent potential new therapeutic targets. We have previously carried out fragment screens using NMR-based activity assays against the uridine preferring nucleoside hydrolase (UNH) and adenosine/guanosine preferring nucleoside hydrolase (AGNH) enzymes. The activities of selected inhibitors, were then validated spectrophotometrically as an orthogonal assay for the determination of both kinetic character and the mode of inhibition. AGNH and UNH kinetic assays were carried out using p-nitrophenyl-β-D-ribofuranoside as the test substrate by measuring the change in absorption at 402 nm, the maximum absorption wavelength for the p-nitrophenol hydrolysis product of the enzyme. The initial rates of absorption were measured against the concentration of inhibitor and substrate in solution. The KI values determined were in general agreement with the IC50 values determined from the NMR-based activity assays. Viramidine was determined to be the most potent inhibitor of AGNH with a KI value of 240 nM. The phenylpyridine fragment hit derivative 3-(4-methylpyridin-3-yl)phenol was determined to be the most potent inhibitor of UNH with a KI value of 3 μM.

Enzyme kinetic assays validate fragment inhibitors of Trichomonas vaginalis nucleoside ribohydrolase enzymes

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.