Abstract
It remains elusive to design peptidomimetic inhibitors of SARS-CoV-2 main protease (Mpro) refractory to multiple deficiencies of Paxlovid (ritonavir-boosted nirmatrelvir), pertaining mainly to E166X mutations-conferred drug resistance and inherent pharmacokinetic limitations to nirmatrelvir. We identify via virtual screening an iso-quinoline P1 moiety in place of the traditional γ-lactam and design iso-quinoline-containing inhibitors with high affinity for Mpro and its nirmatrelvir-resistant E166X mutants. Further optimization at P4 cultivates distinctive peptidomimetic inhibitors with drastically improved pharmacokinetic properties and significantly enhanced antiviral efficacy independent of ritonavir. Two such inhibitors, FD3-32 and FD3-36, also potent against SARS-CoV-1 and MERS-CoV Mpro, are more effective as a monotherapy regimen than Paxlovid in reducing viral loads in vivo and protecting infected male mice from acute lung injury. Here, we report the discovery of next-generation SARS-CoV-2 Mpro inhibitors that overcome the deficiencies of Paxlovid, promising efficacious antivirals critical for mitigating the current and future pandemics of coronaviruses.
Data availability
The PDB accession number for coordinates and structure factors of SARS-CoV-2 Mpro in complex with FD6-31 is 9L13. All other data are available in the main text or the supplementary materials. The initial coordinates and final outputs of MD simulations involved in this study have been deposited in the Zenodo database (https://doi.org/10.5281/zenodo.16948252). Resources, reagents and materials generated in this study are available from the Lead Contact, W.L. (luwuyuan@fudan.edu.cn), upon request. Source data are provided with this paper as Source Data files. Source data are provided in this paper.
Code availability
Input scripts for MD simulations in this study are available in the Zenodo database https://doi.org/10.5281/zenodo.16948252. The codes used for analysis and visualization of chemical space are publicly available on GitHub at https://github.com/LogenLuo/Noncanonical_P1_screening and deposited in Zenodo https://doi.org/10.5281/zenodo.18823333.
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Acknowledgements
We thank Dr. Lingyun Yang in the NMR platform of iHuman Institute, ShanghaiTech University, for his excellent technical assistance on 2D NMR experiments; Prof. Youhua Xie in Shanghai Medical College, Fudan University, for his technical support on virological assays. We also thank the Center for Molecular Modeling and Design, School of Pharmacy, Fudan University, for support of computational resources. We thank the staff of the BL10U2 beamline at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China, for assistance during data collection. This work was supported by grants from the Shanghai Municipal Science and Technology Major Project (ZD2021CY001 to F.W.), the National Natural Science Foundation of China (82030062 to W.L. and 82400683 to J.Z.), the Young Elite Scientists Sponsorship Program by CAST-Doctoral Student Special Plan (G.L.), the China Postdoctoral Science Foundation (2023M740661 to J.Z.), and the National Key Research and Development Program of China (2021YFC2300703 to L.L.).
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W.L., G.L., X.G., and F.W. conceived, supervised, and/or designed the research; G.L. and W.L. designed the inhibitors; G.L., L.F., and J.J.Z. performed or supervised chemical synthesis, purification and structural characterizations; G.W. and M.L. conducted all protein expression and purification in this study; G.L. and G.W. performed 2D-NMR experiments and analyzed spectra; G.L. performed enzymatic inhibition assays and TSA assays; B.Z. and G.W. contributed to crystallization and diffraction data collection. B.Z. determined and analyzed the crystal structure; J.Z. performed in vitro cytotoxicity assays; W.X. and L.L. supervised virological assays in this study; C. L., W.X., G.L., Z.W., J.R., G.W., J.Z., and Y.Y. performed both in vitro and in vivo antiviral assays, as well as related characterizations; G.L. and W.L. wrote the manuscript with assistance of G.W., B.Z., and L.F.
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W.L., G.L., and X.G. are inventors in a patent application (CN202410660959.1; PCT/CN2025/096704) related to compounds in this study. The remaining authors declare no competing interests.
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Luo, G., Wang, G., Liao, C. et al. Next-generation inhibitors of SARS-CoV-2 Mpro overcome the deficiencies of Paxlovid. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71436-6
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DOI: https://doi.org/10.1038/s41467-026-71436-6
