Structural basis for HKU5‑CoV main protease inhibition by the clinical antivirals nirmatrelvir and ensitrelvir

The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a human ACE2-adapted virus highlights the need for antiviral strategies to control emerging HKU5-related merbecoviruses. However, despite the importance of the main protease (M ^pro ) as a key antiviral target, structural and biochemical characterization of HKU5-CoV M ^pro in the context of clinical inhibitors has remained limited. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 M ^pro in its apo state (1.75 Å) and in complex with the approved covalent inhibitor nirmatrelvir (1.91 Å) and the non-covalent inhibitor ensitrelvir (1.55 Å). These structures reveal an induced-fit mechanism, in which the S2 loop containing Met49 adopts an open conformation in the apo state and undergoes inhibitor-specific conformational changes upon nirmatrelvir and ensitrelvir binding. These structures served as a foundation for the characterization of HKU5-CoV-2 M ^pro via modeling and molecular dynamics simulations. Biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 M ^pro exhibited nearly identical kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 M ^pro . Structural analysis revealed a highly conserved S1 subsite but distinct local environments in the S1′, S2, and S4 substrate-binding sites relevant to inhibitor recognition. Despite these variations, nirmatrelvir and ensitrelvir showed potent inhibitory activity, with comparable double-digit nanomolar IC ₅₀ values across all three M ^pro proteins. Computational analyses indicated that HKU5-CoV-2 M ^pro engages nirmatrelvir and ensitrelvir in binding modes comparable to those observed for HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition and providing a structural basis for developing pan-coronavirus antivirals targeting emerging merbecoviruses.