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bioRxiv · 10.64898/2025.12.23.696280

Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates

Saha, D.·Dakhili, S. Y. T.·Mar, E.·Chen, Y. S.·VanPetegem, F.·Bromme, D.
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Abstract

The SARS-CoV-2 main protease (3CLpro) is essential for viral replication and a leading antiviral target. Circulating variants accumulate substitutions on this enzyme, distant from the catalytic site. Surprisingly, mutant enzymes retain full proteolytic activity, though preserved overall activity does not exclude subtler effects on substrate recognition or selectivity, arising from distal structural perturbations. In this study, we compared the steady-state kinetics of wild-type (Wuhan) 3CLpro with enzymes from the Beta (K90R), Lambda (G15S), and Omicron (P132H) variants, using two peptide substrates representing distinct viral polyprotein cleavage sites. All four proteases displayed comparable catalytic efficiencies, similar pH-rate profiles, suggesting conservation of the catalytic mechanism despite sequence variation. The crystal structure of Omicron 3CLpro bound to an Nsp8-Nsp9 peptide revealed a conserved fold and active-site geometry, with the P132H side chain adopting a substrate-dependent conformation that rebuilt its local contacts, indicating towards how a distal substitution is accommodated without perturbing catalysis. Thermal stability measurements identified the sole distinguishing effect of P132H, with Omicron showing altered stability at elevated temperature. A screen of 31 tanshinones against 3CLpro identified T06 with Ki values of 5 uM, as 3CL pro inhibitor. Thus, 3CLpro may tolerate distal substitutions through local structural adaptation, supporting its durability as an antiviral target.

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Saha, D., Dakhili, S. Y. T., Mar, E., Chen, Y. S., VanPetegem, F., Bromme, D.. 2025-12-26. Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates. https://doi.org/10.64898/2025.12.23.696280

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