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.
NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC 50 values, detergent counter screen assays, jumpdilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1 H NMR is shown for the purine-specific enzyme, while 19 F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its K m value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.
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