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

ABSTRACT: 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 (IC₅₀), and relative substrate specificity for 10²-10³ variants. Applied to the SARS-CoV-2 main protease (

SUBMITTER: Aidlen D 

PROVIDER: S-EPMC12873778 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature