2. Academic Validation
  3. Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness

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

  • bioRxiv. 2026 Jan 28:2026.01.27.702130. doi: 10.64898/2026.01.27.702130.
Dylan Aidlen 1,  William Vinh Thanh Vo 2,  Nicholas J Young 3 4,  Julia Rosecrans 5,  Anna Kurianowicz 5,  Shih-Wei Chuo 4,  Garrison P R Asper 6 7,  Dashiell Anderson 4,  Jacob A Posner 6 8,  Duncan F Muir 7,  Nicholas Freitas 7,  Melanie Ott 5 9,  Charles S Craik 4,  Taha Y Taha 5 10,  Margaux M Pinney 6 8 11 9
Affiliations

Affiliations

  • 1 Graduate Program in Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA, USA.
  • 2 Master of Translational Medicine Program, University of California, Berkeley, and University of California, San Francisco, Berkeley, San Francisco, CA, USA.
  • 3 Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, CA, USA.
  • 4 Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
  • 5 Gladstone Institutes, San Francisco, CA, USA.
  • 6 Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.
  • 7 Graduate Program in Biophysics, University of California, San Francisco, San Francisco, CA, USA.
  • 8 California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, USA.
  • 9 Biohub, San Francisco, San Francisco, CA, USA.
  • 10 Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.
  • 11 Department of Chemistry, University of California, Berkeley, Berkeley, USA.
PMID: 41659632 DOI: 10.64898/2026.01.27.702130
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-MEKpro), 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 (Mpro), HT-MEKpro 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.

Figures
Products
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-32718
    99.13%, Src Inhibitor