A Time-Resolved FRET Activity Assay to Distinguish Enzymatic Inhibition from PROTAC-Mediated Degradation of...

Proteolysis-targeting chimeras (PROTACs) are emerging as a promising strategy for antiviral drug development and are mechanistically distinct from classical small-molecule inhibitors. Many PROTAC designs targeting viral proteases, such as the SARS-CoV-2 main protease (M^pro), employ warheads derived from active-site inhibitors, thereby combining proximity-induced degradation with potential enzymatic inhibition. This dual functionality complicates the experimental distinction between degradation-driven effects and direct inhibition of catalytic activity on viral replication. To address this, we established a fluorescence resonance energy transfer (FRET)-based assay to quantify the catalytic activity of recombinant M^pro in the presence of inhibitors and degraders. The assay employs a quenched fluorogenic peptide substrate (MI-2822) containing the canonical M^pro cleavage sequence, flanked by a fluorophore and a quencher. Proteolytic cleavage results in a time-dependent increase in fluorescence, enabling continuous kinetic monitoring of enzyme activity. Recombinant M^pro expressed in Escherichia coli is purified and incubated with the FRET substrate under defined buffer conditions, and fluorescence is recorded over time to derive enzymatic activity profiles. The assay is controlled using the clinically approved active-site inhibitor nirmatrelvir, which efficiently suppresses substrate cleavage. In contrast, PROTACs targeting M^pro outside the catalytic cleft do not substantially reduce the fluorescence increase, indicating that they do not measurably inhibit enzymatic activity under the tested conditions. This protocol provides a robust and reproducible approach to distinguish between enzymatic inhibition and degradation-associated mechanisms. It is broadly applicable for the mechanistic characterization of PROTACs and other bifunctional molecules targeting viral proteases.