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 (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