Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir

Bhandari, Dipendra; Gerlits, Oksana; Keable, Stephen; Coates, Leighton; Aniana, Annie; Ghirlando, Rodolfo; Nashed, Nashaat T.; Kovalevsky, Andrey; Louis, John M.

doi:10.1038/s42003-025-08487-w

Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir

Journal Article · Thu Jul 17 00:00:00 EDT 2025 · Communications Biology

DOI:https://doi.org/10.1038/s42003-025-08487-w· OSTI ID:2573349

Bhandari, Dipendra ^[1]; Gerlits, Oksana ^[2]; ^[1]; ^[1]; Aniana, Annie ^[3]; ^[3]; ^[3]; ^[1]; ^[3]

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Tennessee Wesleyan University, Athens, TN (United States)
National Institutes of Health (NIH), Bethesda, MD (United States)

We investigate the effects of two naturally selected substitution and deletion (Δ) mutations, constituting part of the substrate binding subsites S2 and S4, on the structure, function, and inhibition of SARS CoV-2 main protease. Comparable to wild-type, MPro^D48Y/ΔP168 undergoes N-terminal autoprocessing essential for stable dimer formation and mature-like catalytic activity. The structures are similar, but for an open active site conformation in MPro^D48Y/ΔP168 and increased dynamics of the S2 helix, S5 loop, and the helical domain. Some dimer interface contacts exhibit shorter H bond distances corroborating the ~40-fold enhanced dimerization of the mutant although its thermal sensitivity to unfolding is 8 °C lower, relative to wild-type. ITC reveals a 3- and 5-fold decrease in binding affinity for nirmatrelvir and ensitrelvir, respectively, and similar GC373 affinity, to MPro^D48Y/ΔP168 relative to wild-type. Structural differences in four inhibitor complexes of MPro^D48Y/ΔP168 compared to wild-type are described. Consistent with enhanced dynamics, the S2 helix and S5 loop adopting a more open conformation appears to be a unique feature of MPro^D48Y/ΔP168 both in the inhibitor-free and bound states. Our results suggest that mutational effects are compensated by changes in the conformational dynamics and thereby modulate N-terminal autoprocessing, K_dimer, catalytic efficiency, and inhibitor binding.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2573349

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 8; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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