Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

Chen, X.; Huang, X.; Ma, Q.; Kuzmic, P.; Zhou, B.; Xu, J.; Liu, B.; Jiang, H.; Zhang, W.; Yang, C.; Wu, S.; Huang, J.; Li, H.; Long, C.; Zhao, X.; Xu, H.; Sheng, Y.; Guo, Y.; Niu, C.; Xue, L.; Xu, Y.; Liu, J.; Zhang, T.; Spencer, J.; Deng, W.; Chen, S.-H.; Xiong, X.; Yang, Z.; Zhong, N.

doi:10.1101/2023.03.09.531862

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bioRxiv · 10.1101/2023.03.09.531862

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

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Abstract

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

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Chen, X., Huang, X., Ma, Q., Kuzmic, P., Zhou, B., Xu, J., Liu, B., Jiang, H., Zhang, W., Yang, C., Wu, S., Huang, J., Li, H., Long, C., Zhao, X., Xu, H., Sheng, Y., Guo, Y., Niu, C., Xue, L., Xu, Y., Liu, J., Zhang, T., Spencer, J., Deng, W., Chen, S.-H., Xiong, X., Yang, Z., Zhong, N.. 2023-03-09. Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor. https://doi.org/10.1101/2023.03.09.531862

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