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

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.…”

“…Further, preliminary experiments that used E. coli cells and growth media as a surrogate for T. vaginalis indicated that adenosine appeared to undergo enzymatic reactions in addition to glycosidic bond hydrolysis. 42 We were encouraged to switch to guanosine as substrate to avoid these complications [M. Reily, personal communication]. Guanosine performs comparably to adenosine in benchtop assays, with the caveat that its higher K m value necessitates using twice the substrate concentration.…”

“…In the absence of forodesine, the substrate was about 90% hydrolyzed after 8 h. By contrast, addition of 25 μM forodesine completely blocks substrate hydrolysis, with inhibition tapering off at doses below 5 μM. The in-cell IC 50 value was determined to be 1.4 ± 1.1 μM by fitting the percent reaction as a function of the compound concentration 42 as shown in Figure 8A. This compares to an IC 50 value of 8.4 ± 1.1 μM against the purified enzyme as shown in Figure 8B.…”

“…In-cell reactions were initiated by adding the appropriate amount of substrate stock solutions to 1 mL of cells and then returning the cells to 37 °C. 42 For reactions carried out in fresh media, cells grown overnight were first centrifuged for 5 min at 12,000 rpm and then resuspended in fresh media. For reactions containing inhibitor, the inhibitor (or DMSO control) was added immediately prior to adding substrate.…”

“…Dose−response curve data was collected in triplicate and analyzed as described previously. 42 For each concentration, percent conversion of substrate was determined by comparing the intensity of the substrate peak to that of the zero hour control. Percent reaction was then determined by comparison to the percent conversion of the 8 h control sample.…”