SummaryRhinovirus 3C protease is an attractive target For therapeutic intervention owing to its important role in virion maturation and inFectivity. In order to Facilitate the identification of potential 3C protease inhibitors, we have developed a continuous Fluorescence assay using 5-[(2-aminoethyl}amino]naphthalene-l-sulphonic acid (Edans) as a Fluorescent donor and 4-(4-dimethylaminophenylazo}benzoic acid (Dabcyl) as a quenching acceptor. Several F1uorogenic peptide substrates For 3C protease were synthesized by both solution chemistry and solid phase peptide synthesis. One of the synthetic Edans/ Dabcyl substrates, with an amino acid sequence derived From the 2C/3A site of the virus polyprotein, yielded a 24-Fold increase in Fluorescence intensity after 3C cleavage. Data regarding substrate cleavage kinetics, assay sensitivity and optimization are presented. The application ofthisassay to the evaluation of 3C protease inhibitors is also shown.

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