Abstract: bioRxiv preprint doi: https://doi.org/10.1101/2021.11.28.470226; this version posted November 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Main protease mutants of SARS-CoV-2 variants remain susceptible to PF-07321332 Sven Ullrich, Kasuni B. Ekanayake, Gottfried Otting, Christoph Nitsche* Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia *christoph.nitsche@anu.edu.au Graphical Abstract Keywords SARS-CoV-2, main protease, variants, PF-07321332, inhibitors Abstract The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic PF-07321332. We expressed Mpro of five SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that PF-07321332 has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of specific Mpro inhibitors such as PF-07321332 is not compromised in current COVID-19 variants. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.28.470226; this version posted November 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Since its emergence in late 2019,1 COVID-19 has significantly impacted on societies worldwide.2 More than 5 million deaths have been attributed to COVID-19, with the number of confirmed SARS-CoV-2 infections surpassing 250 million.3 The outbreak of SARS-CoV-2 prompted multiple successful vaccine development campaigns.4 Currently approved vaccines, such as viral vector or mRNA vaccines, successfully limited the pandemic’s impact on global health.5, 6 Most COVID-19 vaccines function by stimulating an immune response against the SARS-CoV-2 spike protein (S)7-9 but, as the spike gene has gathered pronounced genetic variability,10, 11 it is a common concern that the effectiveness of existing vaccines may be affected by those variants of SARS-CoV-2.5, 6, 10, 12 At the time of writing, the World Health Organization (WHO) lists four variants of concern (VOC; Alpha, Beta, Gamma, Delta) and two variants of interest (VOI; Lambda, Mu).13 A possible adjustment of the vaccines to currently circulating lineages of SARS-CoV-2 is being investigated.14-16 It is clear that the deployment of vaccines remains the best public health measure to control the spread of SARSCoV-2 and the severe health effects of COVID-19.17, 18 Complementary to preventive vaccines, antiviral drugs are urgently needed to combat COVID-19.19 Since the discovery of SARS-CoV-1 in 2003,20 several coronaviral drug targets have been identified,21..