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

The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a potential zoonotic threat, owing to its adaptation to the human angiotensin-converting enzyme 2 receptor, highlights the need for antiviral strategies to control emerging HKU5-CoVs. 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. In this study, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro in its apo state and in complex with the clinical inhibitors nirmatrelvir and ensitrelvir. 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. Integrated structural modeling and molecular dynamics simulations showed that HKU5-CoV-2 Mpro retains the ligand-induced active-site rearrangements observed in HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition. These findings provide a structural framework for understanding the susceptibility of emerging Merbecoviruses to clinical Mpro inhibitors and support the development of pan-Coronavirus antivirals. Author summaryAs coronaviruses continue to emerge from wildlife reservoirs, determining whether current clinical antivirals can inhibit divergent viral targets and how they engage these proteins is crucial. This study focuses on Pipistrellus bat coronavirus HKU5, particularly the newly identified lineage 2 (HKU5-CoV-2), which has recently attracted attention as a potential zoonotic coronavirus. We determined high-resolution crystal structures of the HKU5-CoV-1 main protease and used these structures to build and analyze models of the HKU5-CoV-2 protease. Our biochemical and structural analyses show that approved COVID-19 protease inhibitors, including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova), potently inhibit HKU5 Mpro and reveal conserved features of inhibitor recognition. These findings provide a structural foundation for designing coronavirus protease inhibitors with broader activity against emerging coronaviruses.