Structural basis for the inhibition of HKU8 main protease by nirmatrelvir and ensitrelvir

The wide distribution and high divergence of zoonotic coronaviruses (CoVs) poses a continuous threat to public health. Some CoVs, such as HKU8, harbor a high potential to breach the species barrier and emerge in human populations. However, whether the current antivirals are effective against HKU8 CoV remains poorly understood. Here, we studied the effectiveness of two clinically approved antivirals, nirmatrelvir and ensitrelvir, against the main protease (M^pro) of the HKU8. Both nirmatrelvir and ensitrelvir can inhibit HKU8 M^pro, but their efficacy is attenuated by approximately 5-fold and 15-fold, respectively, compared to their activity against SARS-CoV-2 M^pro. To elucidate the structural basis of this reduced potency, crystal structures of HKU8 M^pro in complex with nirmatrelvir and ensitrelvir were determined at 2.43 Å and 1.70 Å resolution, respectively. The HKU8 M^pro features a proline at position 188 instead of the characteristic 3₁₀-helix found in beta-CoVs. This difference results in a reduced volume of this subsite, which may lead to suboptimal binding of both inhibitors and reduced efficacy. These findings provide crucial information on the effectiveness of M^pro-targeting antivirals cross different species and highlight the S2 subsite as a key determinant for their activity. It may help the design of next-generation broad-spectrum antivirals against CoVs.