Abstract
Herein we report the development of novel covalent inhibitors of the SARS-CoV-2 main protease (Mpro). The developed compounds VPC285785 and VPC285786 demonstrated moderate inhibition of Mpro (IC50 0.8 µM vs. Nirmatrelvir 0.03 µM), whereas VPC285786 additionally inhibited human cathepsin L (CatL; IC50 4.2 µM vs Nirmatrelvir > 100 µM). In vitro metabolic stability studies in human and mouse microsomes revealed that VPC285786 demonstrated enhanced metabolic stability compared to Nirmatrelvir, with minimal turnover observed during the experimental window. Subsequent mass spectrometry analysis identified putative metabolic products consistent with previously reported oxidation patterns. Pharmacokinetic studies in mice demonstrated that VPC285785 achieved 15% oral bioavailability, supporting the potential for oral administration, whereas VPC285786 showed limited oral exposure despite superior metabolic stability. A side-by-side efficacy study of VPC285785 and Nirmatrelvir in a Murine Hepatitis Virus (MHV) infection model demonstrated that VPC285785 significantly reduced viral load in liver, brain, and spleen tissues compared to vehicle- and Nirmatrelvir-treated controls, while maintaining healthy liver function parameters. These results lay the foundation for further development of VPC285785-series antivirals that could be used as oral, single-agent therapies for SARS-CoV-2 infection, particularly given their dual-targeting mechanism and favorable toxicity profile.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Chew, K. W., Malani, P. N. & Gandhi, R. T. COVID-19 therapeutics for nonhospitalized patients: Updates and future directions. JAMA 330, 1519–1520 (2023).
Hammond, J. et al. Oral nirmatrelvir for high-risk, nonhospitalized adults with Covid-19. N Engl J Med 386, 1397–1408 (2022).
Hammond, et al. Nirmatrelvir for vaccinated or unvaccinated adult outpatients with Covid-19. New England J. Med. 390(13), 1186–1195 (2024).
Foisy, M. M., Yakiwchuk, E. M. & Hughes, C. A. Induction effects of ritonavir: implications for drug interactions. Ann. Pharmacother. 42, 1048–1059 (2008).
Kis, O., Robillard, K., Chan, G. N. & Bendayan, R. The complexities of antiretroviral drug-drug interactions: Role of ABC and SLC transporters. Trends Pharmacol Sci 31, 22–35 (2010).
Casey, B. 3rd., Vernick, R. C., Bahekar, A., Patel, D. & Ncogo Alene, I. Ranolazine Toxicity Secondary to Paxlovid. Cureus 15, e37153 (2023).
Shen, D., Gong, Y., Qian, Y., Zhu, J. & Gao, J. Nirmatrelvir/ritonavir treatment of patients with COVID-19 taking tacrolimus: Case series describing the results of drug–drug interactions. J. Int. Med. Res. 52(5), 03000605241247705 (2024).
Michael, S., Heilbronner, R., Lloyd, C. M. & Levitin, H. W. Paxlovid-induced tacrolimus toxicity in the treatment of COVID-19: A case report. Cureus 15, e35489 (2023).
Kwon, E.-J. et al. Treatment of acute tacrolimus toxicity with phenytoin after Paxlovid (nirmatrelvir/ritonavir) administration in a kidney transplant recipient. Kidney Res Clin Pract 41, 768–770 (2022).
Pérez-Vargas, J. et al. A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants. Emerg Microbes Infect 12, 2246594 (2023).
Xiong, M., Su, H., Zhao, W., Xie, H. & Shao, Q. What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design. Med Res Rev 41, 1965–1998 (2021).
Amorim, VMd. F. et al. 3-Chymotrypsin-like protease (3CLpro) of SARS-CoV-2: Validation as a molecular target, proposal of a novel catalytic mechanism, and inhibitors in preclinical and clinical trials. Viruses 16, 844 (2024).
Liu, T., Luo, S., Libby, P. & Shi, G.-P. Cathepsin L-selective inhibitors: A potentially promising treatment for COVID-19 patients. Pharmacol. Ther. 213, 107587 (2020).
Owen, D. R. et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586–1593 (2021).
Sabbah, D. A., Hajjo, R., Bardaweel, S. K. & Zhong, H. A. An updated review on betacoronavirus viral entry inhibitors: Learning from past discoveries to advance COVID-19 drug discovery. Curr Top Med Chem 21, 571–596 (2021).
Körner, R. W., Majjouti, M., Alcazar, M. A. A. & Mahabir, E. Of mice and men: The coronavirus MHV and Mouse models as a translational approach to understand SARS-CoV-2. Viruses 12, 880 (2020).
Yang, Z. et al. Coronavirus MHV-A59 infects the lung and causes severe pneumonia in C57BL/6 mice. Virologica Sinica 29, 393–402 (2014).
Arévalo, A. P. et al. Ivermectin reduces in vivo coronavirus infection in a mouse experimental model. Sci. Rep. 11, 7132 (2021).
Chaves, I.d.M. et al. Broad-Spectrum Antiviral Efficacy of 7-Deaza-7-Fluoro-2’-C-Methyladenosine Against Multiple Coronaviruses In Vitro and In Vivo. bioRxiv, 2025.01.02.631052 (2025).
Shuipys, T. & Montazeri, N. Optimized protocols for the propagation and quantification of infectious murine hepatitis virus (MHV-A59) Using NCTC clone 1469 and 929 cells. Methods Protoc. 5(1), 5 (2022).
Sonia, S., Aman, K. & Himanshu, S. In-vitro and In-vivo experimental models for MERS-CoV, SARSCoV, and SARS-CoV-2 viral infection: A compendious review. Recent Pat. Biotechnol. 16, 82–101 (2022).
Bai, B. et al. Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors. RSC Med. Chem. 12, 1722–1730 (2021).
Yuan, Z. et al. Site-selective, late-stage C−H 18F-fluorination on unprotected peptides for positron emission tomography imaging. Angew. Chem. Int. Ed. 57, 12733–12736 (2018).
Lee, A. J. et al. A 14C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals β-hydroxy-β-methylbutyrate as a metabolite. Amino Acids 47, 917–924 (2015).
Kumari, A. & Singh, R. K. Medicinal chemistry of indole derivatives: Current to future therapeutic prospectives. Bioorg. Chem. 89, 103021 (2019).
Norwood, V. M. IV. & Huigens, R. W. III. Harnessing the chemistry of the indole heterocycle to drive discoveries in biology and medicine. ChemBioChem 20, 2273–2297 (2019).
Thanikachalam, P. V., Maurya, R. K., Garg, V. & Monga, V. An insight into the medicinal perspective of synthetic analogs of indole: A review. Eur. J. Med. Chem. 180, 562–612 (2019).
Sheikhi, N., Bahraminejad, M., Saeedi, M. & Mirfazli, S. S. A review: FDA-approved fluorine-containing small molecules from 2015 to 2022. Eur. J. Med. Chem. 260, 115758 (2023).
Meanwell, N. A. Fluorine and fluorinated motifs in the design and application of bioisosteres for drug design. J. Med. Chem. 61, 5822–5880 (2018).
Inoue, M., Sumii, Y. & Shibata, N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega 5, 10633–10640 (2020).
Weiss, D. R. et al. Balanced permeability index: A multiparameter index for improved in vitro permeability. ACS Med. Chem. Lett. 15, 457–462 (2024).
Spielvogel, C. P. et al. Enhancing blood-brain barrier penetration prediction by machine learning-based integration of novel and existing, in silico and experimental molecular parameters from a standardized database. J. Chem. Inf. Model. 65, 2773–2784 (2025).
Wager, T. T., Hou, X., Verhoest, P. R. & Villalobos, A. Central nervous system multiparameter optimization desirability: Application in drug discovery. ACS Chem. Neurosci. 7, 767–775 (2016).
Levterov, V. V. et al. 2-Oxabicyclo[2.2.2]octane as a new bioisostere of the phenyl ring. Nat. Commun. 14, 5608 (2023).
de Sena, M. et al. The Use of conformational restriction in medicinal chemistry. Current Topics Med. Chem. 19(19), 1712–1733 (2019).
Yan, S. & Wu, G. Potential 3-chymotrypsin-like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID-19 vaccine and drug development. FASEB J. 35, e21573 (2021).
Huang, Y., Yang, C., Xu, X.-F., Xu, W. & Liu, S.-W. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol. Sin. 41, 1141–1149 (2020).
Eng, H. et al. Disposition of nirmatrelvir, an orally bioavailable inhibitor of SARS-CoV-2 3C-Like Protease, across animals and humans. Drug Metab. Dispos. 50, 576–590 (2022).
Toro, A. et al. Blood matters: the hematological signatures of Coronavirus infection. Cell Death Dis. 15, 863 (2024).
Acknowledgements
We thank Drs. Jaeyong Lee and Shoeib Moradi from the lab of Professor Natalie Strynadka (UBC Life Sciences Institute) for providing recombinant Mpro protein. This work was supported by grants from the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding program of the Canadian Institutes of Health Research (CIHR) [VR3-172639 (AC and RNY) and OV3-1760631 (AC). Operating funds were provided by Canada Foundation for Innovation (CFI) in collaboration with the British Columbia Knowledge Development Fund (BCKDF) #36194 (AC), and the Canada Research Chairs Program (CRC-2020-00007, AC). MC and APA received funding from FOCEM—Fondo para la Convergencia Estructural del Mercosur (https://focem.mercosur.int/es/) (Grant Number: COF 03/11). MC is a fellow of Sistema Nacional de Investigadores, Agencia Nacional de Investigación e Innovación, Uruguay. (https://www.anii.org.uy/).
Author information
Authors and Affiliations
Contributions
Designed the study: J.R.S., A.T., M.C., G.G., R.N.Y., A.C. Performed experiments, analyzed data: J.R.S., A.T., A.S., S.K., F.B., A.P.A. Obtained funding: M.C., R.N.Y., A.C. Provided supervision M.C., G.G., R.N.Y., A.C. Wrote the manuscript: J.R.S., A.T. All authors edited and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
RNY is founder and president of Mesentech which is not affiliated with this research. RNY and AC are members of the scientific advisory board of Variational AI, Inc. which is not affiliated with this research. All other authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Smith, J.R., Toro, A., Sabater, A. et al. Pharmacokinetics, pathology and efficacy of SARS-CoV-2 main protease inhibitor VPC285785 in a murine model of coronavirus infection. Sci Rep (2026). https://doi.org/10.1038/s41598-026-36842-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-36842-2
