Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness

Proteases govern essential biological processes and are key drug targets, yet how protease sequence variation quantitatively reshapes biochemical parameters and constrains biological fitness remains poorly understood. Here, we integrate high-throughput in vitro enzymology with cellular assays to link protease sequence, biochemistry, and fitness. We extend a microfluidic platform for high-throughput protease enzymology (HT-MEK pro ), which is broadly applicable across protease families and catalytic classes, enabling measurement of catalytic turnover ( k cat ), Michaelis constant ( K M ), inhibitor potency (IC 50 ), and relative substrate specificity for 10 2 -10 3 variants. Applied to the SARS-CoV-2 main protease (M pro ), HT-MEK pro generated parallel catalytic and inhibitory landscapes for >400 variants. Integration with viral replication and in-cell cleavage assays reveals that variants with altered substrate specificity fail to support replication, suggesting imbalanced polyprotein processing as a constraint on viral fitness. More broadly, these data can enable mechanistically grounded modeling of protease sequence-property relationships and inform strategies for pharmacological modulation beyond active-site inhibition.