NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases.

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 IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F 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 Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

Ligand-Efficient Inhibitors of Trichomonas vaginalis Adenosine/Guanosine Preferring Nucleoside Ribohydrolase.

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis and is the most prevalent, nonviral sexually transmitted disease. The parasite has shown increasing resistance to the current 5-nitroimidazole therapies indicating the need for new therapies with different mechanisms. T. vaginalis is an obligate parasite that scavenges nucleosides from host cells and then uses salvage pathway enzymes to obtain the nucleobases. The adenosine/guanosine preferring nucleoside ribohydrolase was screened against a 2000-compound diversity fragment library using a 1H NMR-based activity assay. Three classes of inhibitors with more than five representatives were identified: bis-aryl phenols, amino bicyclic pyrimidines, and aryl acetamides. Among the active fragments were 10 compounds with ligand efficiency values greater than 0.5, including five with IC50 values <10 µM. Jump-dilution and detergent counter screens validated reversible, target-specific activity. The data reveals an emerging SAR that is guiding our medicinal chemistry efforts aimed at discovering compounds with nanomolar potency.