Structural Basis for Inhibition of the HKU5-CoV Main Protease by Clinical SARS-CoV-2 Protease Inhibitors

Abstract

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 (Mpro) as a key antiviral target, structural and biochemical characterization of HKU5-CoV Mpro in the context of clinical inhibitors has remained limited. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro in its apo state (1.75 [A]) and in complex with the approved covalent inhibitor nirmatrelvir (1.91 [A]) and the non-covalent inhibitor ensitrelvir (1.55 [A]). 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 Mpro via modeling and molecular dynamics simulations. Biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 Mpro exhibited nearly identical kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 Mpro. 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 IC50 values across all three Mpro proteins. Computational analyses indicated that HKU5-CoV-2 Mpro 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.