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. With no specific antivirals approved for merbecoviruses, repurposing SARS-CoV-2 Mpro inhibitors is a promising strategy. However, their inhibitory activity and binding mechanisms against HKU5-CoV Mpro remain poorly understood. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro 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 Leu49 adopts an open conformation in the apo state and undergoes inhibitor-specific conformational changes upon inhibitor binding. These structures provided a foundation for characterizing HKU5-CoV-2 Mpro via modeling and molecular dynamics simulations, demonstrating conserved inhibitor-binding modes across both lineages. Consistent with this, biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 Mpro exhibited nearly identical enzyme kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 Mpro. Structural comparison of the HKU5-CoV-1 and SARS-CoV-2 Mpro revealed a conserved S1 subsite, alongside key variations: Met25 lining S1', a flexible Leu49-containing S2 loop, and a Ser171-mediated expanded S4 cavity. 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 HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition and providing a structural basis for developing pan-coronavirus antivirals targeting emerging merbecoviruses.