Protocol assesses fluorescence from AMC in screening assays for serine protease inhibitors, indicating efficacy.
Purpose: performing single point test of selected compounds from screening libraries containing serine-targeting fragments and serine protease inhibitors against DENV NS2B-NS3 to find warheads and candidates for medicinal optimization General description: This protocol details the fluorescent assays for ZIKV NS2B-NS3 cleavage of substrate Bz-Nle-KRR-AMC peptide. This method measures the fluorescence from the released AMC product as a result of the enzymatic reaction. When hydrolyzed, AMC is liberated from the peptide substrate. Excitation of AMC at 350 nm emits a resonant energy of 450 nm. When the enzyme is inhibited, the fluorescence signal will decrease as a result of lower enzyme activity. The screening method is validated by calculating the Z prime number of each plate, and the percentage of inhibition is calculated and then evaluated for inhibitory efficacy. Outcome: hits were selected from screening assays with more than 50% inhibition
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Barr et al. (2024) studied this question.