Discovery of Ligand-Efficient Scaffolds for the Design of Novel Trichomonas vaginalis Uridine Nucleoside Ribohydrolase Inhibitors Using Fragment Screening

Abstract

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis. The increasing prevalence of strains resistant to the current 5-nitroimidazole treatments creates the need for novel therapies. T. vaginalis cannot synthesize purine and pyrimidine rings and requires salvage pathway enzymes to obtain them from host nucleosides. The uridine nucleoside ribohydrolase was screened using an 19F NMR-based activity assay against a 2000-compound fragment diversity library. Several series of inhibitors were identified including scaffolds based on acetamides, cyclic ureas or ureas, pyridines, and pyrrolidines. A number of potent singleton compounds were identified, as well. Eighteen compounds with IC50 values of 20 μM or lower were identified, including some with ligand efficiency values of 0.5 or greater. Detergent and jump-dilution counter screens validated all scaffold classes as target-specific, reversible inhibitors. Identified scaffolds differ substantially from 5-nitroimidazoles. Medicinal chemistry using the structure–activity relationship emerging from the fragment hits is being pursued to discover nanomolar inhibitors.

Direct Measurement of Nucleoside Ribohydrolase Enzyme Activities in Trichomonas vaginalis Cells Using 19F and 13C-Edited 1H NMR Spectroscopy

“…This assay is used routinely with purified UNH in buffered solution to screen compounds and compound mixtures. 18,21,42 The lack of fluorine signals from buffer solution or test compounds greatly simplifies spectral analysis. This spectral "editing" translated directly to in-cell assays as shown in Figure 1A.…”

“…18,19 Fragment screening of both enzymes identified at least three classes of compounds with ∼100 μM potency and ligand efficiency > 0.5, with the distinct active sites resulting in chemically distinct classes of fragment inhibitors. 20,21 Structure-guided medicinal chemistry is now being used to improve the potency of these fragment leads. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents.…”

“…Purine-specific (AGNH) and pyrimidine-specific (UNH) nucleoside ribohydrolases from T. vaginalis have been previously characterized by us to be distinct, druggable antitrichomonal targets. , Fragment screening of both enzymes identified at least three classes of compounds with ∼100 μM potency and ligand efficiency > 0.5, with the distinct active sites resulting in chemically distinct classes of fragment inhibitors. , Structure-guided medicinal chemistry is now being used to improve the potency of these fragment leads.…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“… 79 After following up on several hits and concluding the target, UNH, was druggable, a fragment screen using a 2000-member library in mixtures of six fragments was employed to identify diverse chemical scaffolds that would be suitable for drug discovery efforts. 80 Mixtures with at least 75% inhibition were deconvoluted and 97 hits were identified (4.9% hit rate). 18 compounds had an IC 50 under 20 μM, several of which had a LE above 0.5, which is ideal for elaboration.…”

“… 81 In particular, a 3-hydroxypyrrolidine was further investigated as a fragment scaffold that could be expanded due to its potency, LE, and vectors for SAR exploration. 80 …”