Assay for Screening of Compounds that Inhibit Enzymatic Activity of EV-A71 2Apro v1

This protocol describes a biochemical assay used in screening to identify compounds that inhibit the enzymatic activity of the EV-A71 2A protease. Under standard conditions, the enzyme cleaves its substrate (DABCYL-RDKITTLGKFGQDE-EDANS), releasing the fluorogenic moiety EDANS, increasing fluorescence. Prior to cleavage, fluorescence is quenched by the DABCYL moiety. In the presence of compounds that inhibit the enzyme’s activity, the substrate remains uncleaved, resulting in a low fluorescent signal. This enables the determination of % inhibition at a given compound concentration, and allows for subsequent IC₅₀ determination when a dose-response format is applied. This protocol was established through the transfer and adaptation of an assay originally developed at the Weizmann Institute of Science, as a part of the ASAP Discovery Consortium.