Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

Abstract

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells. The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells. New viral infectious diseases are emerging and have caused major public health crises in recent years. Reported examples in the last 20 years include severe acute respiratory syndrome coronavirus (SARS-CoV), H1N1 influenza, the Middle East respiratory syndrome coronavirus, Ebola virus disease, and Zika virus (1Majumder J. Minko T. Recent developments on therapeutic and diagnostic approaches for COVID-19.AAPS J. 2021; 23: 14Crossref PubMed Scopus (125) Google Scholar). The world continues to grapple with a global pandemic caused by a novel coronavirus, SARS-CoV-2 that was initially reported to the World Health Organization (WHO) on December 31, 2019 (2Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu B. J. with a coronavirus of PubMed Scopus Google Scholar). the virus as the severe acute respiratory syndrome to with the at the of the viral as a public health of concern of the on of severe acute respiratory and PubMed Scopus Google and as a of SARS-CoV-2 have emerging and the world the of the COVID-19 SARS-CoV-2 is and each virus with a is a The as well as public health variants that COVID-19 for to global public health and as variants variants of interest variants of concern and variants of of the has and and have as is a infectious coronavirus that be in COVID-19 have for have to be or be as to be to coronavirus 2019 L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google main protease to as is a protease that is for the of the and by the SARS-CoV-2 The protease these that are for viral L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). to in viral of SARS-CoV-2 Mpro for the of We have reported on the and efficacy of nirmatrelvir SARS-CoV-2 Mpro inhibitor with in vitro coronavirus activity with and in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). Nirmatrelvir has activity in a SARS-CoV-2 and has plasma concentrations that the in vitro in a in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). we on the catalytic activity of observed Mpro mutations in SARS-CoV-2 VOC/VOI, in vitro efficacy of nirmatrelvir against these mutant the solution-phase structural and of nirmatrelvir bound to the Mpros from β, λ, and wildtype Mpro from the original Washington variant and SARS-CoV-2 Mpro were and to as by a with a of for a of each and the and P132H Mpro to be by Mpro assay was to enzyme catalytic activity by of the at varying concentrations L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google The and were for the and P132H Mpro The catalytic of the and P132H are similar to that of wildtype Mpro These data suggest that the and P132H Mpro variants comparable enzymatic to wildtype we evaluated the of nirmatrelvir to wildtype and and P132H Mpro enzymatic Nirmatrelvir wildtype Ki of and the the and P132H Mpro of the enzyme potency to that of wildtype Mpro by the of Ki with and that the Ki are for the and P132H Mpro was a in potency for Mpro observed in the Lambda variant to wildtype catalytic for SARS-CoV-2 Mpro enzyme in a vitro potency of nirmatrelvir for the SARS-CoV-2 mutant Mpro activity in a as a for Ki compared to wildtype. to wildtype the of Ki to the and is than the to that are are from the of Ki to the and is than the to that are are from as a for Ki compared to The the of Ki to the and is than the to that are are from in a The of nirmatrelvir bound to the were to (K90R), (G15S), and of each of these mutant with the wildtype the of was reported in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google J. P. Yang Zhang B. Zhang J. basis of main of coronavirus bound to 2021; Scopus Google that the of nirmatrelvir is by the with the the observed in the wildtype as in these mutations are to the with Å to from the and Å and to from the The that the mutations to of the the or the of the characterization of nirmatrelvir bound to Mpro of the of nirmatrelvir bound to wildtype SARS-CoV-2 Mpro and Mpro P132H and and and and of the on the the and of the mutant in for the are from variant of Mpro structural the solution-phase structural dynamics of and P132H with the wildtype Mpro were wildtype and mutant in the dynamics of or P132H from wildtype These suggest that the mutations a from wildtype solution-phase wildtype and SARS-CoV-2 Mpro evaluated the catalytic activity and potency of nirmatrelvir against the Mpro mutations observed in the prevalent Alpha (α, B.1.1.7), Beta (β, B.1.351), and Gamma (γ, P.1), a Lambda (λ, with a and Omicron (ο, with a P132H The and P132H Mpro mutations comparable enzymatic as compared to wildtype Mpro as by catalytic We that nirmatrelvir has a comparable potency against these mutant Mpros to wildtype from the original Washington of and P132H to wildtype Mpro and in In with from the of solution-phase of solution-phase dynamics for and P132H to wildtype and for from in the the of nirmatrelvir bound to and P132H Mpro variants that is the of nirmatrelvir to the wildtype Mpro the of the is by the in with the solution-phase These observations are with reported biochemical characterization with similar and nirmatrelvir potency protease of SARS-CoV-2 variants to PubMed Scopus Google Hu Zhang Wang J. The P132H in the main protease of Omicron SARS-CoV-2 or PubMed Scopus Google Scholar). Nirmatrelvir to in vitro efficacy against Omicron to wildtype L. W. P. B. J. P. J. and Nirmatrelvir active against SARS-CoV-2 Omicron and variants of PubMed Scopus Google L. B. Zhu active Mpro is a inhibitor of SARS-CoV-2 variants of PubMed Google and maintain in vitro activity against the SARS-CoV-2 Omicron Google P. Wang SARS-CoV-2 Omicron variant is to and the PubMed Scopus Google of SARS-CoV-2 variants to and the potential for pandemic to be is to emerging to potential to the efficacy of and the of mutations to of protease and the in vitro activity of nirmatrelvir against the and Omicron variants of Mpro and the structural basis for of in vitro potency against these mutant the for activity against variants mutations in the Mpro and the wildtype Mpro was on J. Hu W. Zhang in vitro of and PubMed Scopus Google Scholar). protease was and the was to each of the variants or The was for of was with of and at of was The was a of and were in 20 with a and the was by at The was by a a by with and a to Mpro and characterization and enzymatic activity of the main protease Mpro of SARS-CoV-2 wildtype and variants was a assay L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google Scholar). The SARS-CoV-2 Mpro assay the activity of SARS-CoV-2 Mpro protease to a with the on a The of the is a on a The assay 20 nM enzyme to nM SARS-CoV-2 Mpro mutant protease was to and enzyme were with the of were from with the to activity from a of enzyme to nM SARS-CoV-2 Mpro mutant protease was to assay in a that in a with and were to for 20 of to to the activity was from the and as activity on activity and enzyme Ki were to the with the enzyme to the to the for each and the to and of SARS-CoV-2 Mpro were in at was with well 20 to and to were at and in was to the and to the for at were a of well and in in for data data were at at of the at a of of for the of the of the at the was by the of the a with The were by and the data as for wildtype SARS-CoV-2 Mpro in with L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). data and for each of the mutant Mpro are in for γ β γ β γ β of in to the in to the in to the in to the in to the in to the as by and for and are the observed and is the as for of the data from in in to the the of the as a for Scopus Google as by and P. and data PubMed Scopus Google and are the observed and in a was at and and with protease and to for of were on the of of in in a The and have with the (K90R), (G15S), and (P132H). New viral infectious diseases are emerging and have caused major public health crises in recent years. Reported examples in the last 20 years include severe acute respiratory syndrome coronavirus (SARS-CoV), H1N1 influenza, the Middle East respiratory syndrome coronavirus, Ebola virus disease, and Zika virus (1Majumder J. Minko T. Recent developments on therapeutic and diagnostic approaches for COVID-19.AAPS J. 2021; 23: 14Crossref PubMed Scopus (125) Google Scholar). The world continues to grapple with a global pandemic caused by a novel coronavirus, SARS-CoV-2 that was initially reported to the World Health Organization (WHO) on December 31, 2019 (2Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu B. J. with a coronavirus of PubMed Scopus Google Scholar). the virus as the severe acute respiratory syndrome to with the at the of the viral as a public health of concern of the on of severe acute respiratory and PubMed Scopus Google and as a of SARS-CoV-2 have emerging and the world the of the COVID-19 SARS-CoV-2 is and each virus with a is a The as well as public health variants that COVID-19 for to global public health and as variants variants of interest variants of concern and variants of of the has and and have as SARS-CoV-2 is a infectious coronavirus that be in COVID-19 have for have to be or be as to be to coronavirus 2019 L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). SARS-CoV-2 main protease to as is a protease that is for the of the and by the SARS-CoV-2 The protease these that are for viral L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). to in viral of SARS-CoV-2 Mpro for the of We have reported on the and efficacy of nirmatrelvir SARS-CoV-2 Mpro inhibitor with in vitro coronavirus activity with and in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). Nirmatrelvir has activity in a SARS-CoV-2 and has plasma concentrations that the in vitro in a in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). we on the catalytic activity of observed Mpro mutations in SARS-CoV-2 VOC/VOI, in vitro efficacy of nirmatrelvir against these mutant the solution-phase structural and of nirmatrelvir bound to the Mpros from β, λ, and wildtype Mpro from the original Washington variant and SARS-CoV-2 Mpro were and to as by a with a of for a of each and the and P132H Mpro to be by Mpro assay was to enzyme catalytic activity by of the at varying concentrations L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google The and were for the and P132H Mpro The catalytic of the and P132H are similar to that of wildtype Mpro These data suggest that the and P132H Mpro variants comparable enzymatic to wildtype we evaluated the of nirmatrelvir to wildtype and and P132H Mpro enzymatic Nirmatrelvir wildtype Ki of and the the and P132H Mpro of the enzyme potency to that of wildtype Mpro by the of Ki with and that the Ki are for the and P132H Mpro was a in potency for Mpro observed in the Lambda variant to wildtype catalytic for SARS-CoV-2 Mpro enzyme in a vitro potency of nirmatrelvir for the SARS-CoV-2 mutant Mpro activity in a as a for Ki compared to wildtype. to wildtype the of Ki to the and is than the to that are are from the of Ki to the and is than the to that are are from as a for Ki compared to The the of Ki to the and is than the to that are are from in a The of nirmatrelvir bound to the were to (K90R), (G15S), and of each of these mutant with the wildtype the of was reported in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google J. P. Yang Zhang B. Zhang J. basis of main of coronavirus bound to 2021; Scopus Google that the of nirmatrelvir is by the with the the observed in the wildtype as in these mutations are to the with Å to from the and Å and to from the The that the mutations to of the the or the of the Mpro structural the solution-phase structural dynamics of and P132H with the wildtype Mpro were wildtype and mutant in the dynamics of or P132H from wildtype These suggest that the mutations a from wildtype solution-phase wildtype and SARS-CoV-2 Mpro wildtype Mpro from the original Washington variant and SARS-CoV-2 Mpro were and to as by a with a of for a of each and the and P132H Mpro to be by Mpro assay was to enzyme catalytic activity by of the at varying concentrations L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google The and were for the and P132H Mpro The catalytic of the and P132H are similar to that of wildtype Mpro These data suggest that the and P132H Mpro variants comparable enzymatic to wildtype we evaluated the of nirmatrelvir to wildtype and and P132H Mpro enzymatic Nirmatrelvir wildtype Ki of and the the and P132H Mpro of the enzyme potency to that of wildtype Mpro by the of Ki with and that the Ki are for the and P132H Mpro was a in potency for Mpro observed in the Lambda variant to wildtype The of nirmatrelvir bound to the were to (K90R), (G15S), and of each of these mutant with the wildtype the of was reported in L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google J. P. Yang Zhang B. Zhang J. basis of main of coronavirus bound to 2021; Scopus Google that the of nirmatrelvir is by the with the the observed in the wildtype as in these mutations are to the with Å to from the and Å and to from the The that the mutations to of the the or the of the Mpro structural the solution-phase structural dynamics of and P132H with the wildtype Mpro were wildtype and mutant in the dynamics of or P132H from wildtype These suggest that the mutations a from wildtype solution-phase evaluated the catalytic activity and potency of nirmatrelvir against the Mpro mutations observed in the prevalent Alpha (α, B.1.1.7), Beta (β, B.1.351), and Gamma (γ, P.1), a Lambda (λ, with a and Omicron (ο, with a P132H The and P132H Mpro mutations comparable enzymatic as compared to wildtype Mpro as by catalytic We that nirmatrelvir has a comparable potency against these mutant Mpros to wildtype from the original Washington of and P132H to wildtype Mpro and in In with from the of solution-phase of solution-phase dynamics for and P132H to wildtype and for from in the the of nirmatrelvir bound to and P132H Mpro variants that is the of nirmatrelvir to the wildtype Mpro the of the is by the in with the solution-phase These observations are with reported biochemical characterization with similar and nirmatrelvir potency protease of SARS-CoV-2 variants to PubMed Scopus Google Hu Zhang Wang J. The P132H in the main protease of Omicron SARS-CoV-2 or PubMed Scopus Google Scholar). Nirmatrelvir to in vitro efficacy against Omicron to wildtype L. W. P. B. J. P. J. and Nirmatrelvir active against SARS-CoV-2 Omicron and variants of PubMed Scopus Google L. B. Zhu active Mpro is a inhibitor of SARS-CoV-2 variants of PubMed Google and maintain in vitro activity against the SARS-CoV-2 Omicron Google P. Wang SARS-CoV-2 Omicron variant is to and the PubMed Scopus Google of SARS-CoV-2 variants to and the potential for pandemic to be is to emerging to potential to the efficacy of and the of mutations to of protease and the in vitro activity of nirmatrelvir against the and Omicron variants of Mpro and the structural basis for of in vitro potency against these mutant the for activity against variants mutations in the Mpro We evaluated the catalytic activity and potency of nirmatrelvir against the Mpro mutations observed in the prevalent Alpha (α, B.1.1.7), Beta (β, B.1.351), and Gamma (γ, P.1), a Lambda (λ, with a and Omicron (ο, with a P132H The and P132H Mpro mutations comparable enzymatic as compared to wildtype Mpro as by catalytic We that nirmatrelvir has a comparable potency against these mutant Mpros to wildtype from the original Washington of and P132H to wildtype Mpro and in In with from the of solution-phase of solution-phase dynamics for and P132H to wildtype and for from in the the of nirmatrelvir bound to and P132H Mpro variants that is the of nirmatrelvir to the wildtype Mpro the of the is by the in with the solution-phase These observations are with reported biochemical characterization with similar and nirmatrelvir potency protease of SARS-CoV-2 variants to PubMed Scopus Google Hu Zhang Wang J. The P132H in the main protease of Omicron SARS-CoV-2 or PubMed Scopus Google Scholar). Nirmatrelvir to in vitro efficacy against Omicron to wildtype L. W. P. B. J. P. J. and Nirmatrelvir active against SARS-CoV-2 Omicron and variants of PubMed Scopus Google L. B. Zhu active Mpro is a inhibitor of SARS-CoV-2 variants of PubMed Google and maintain in vitro activity against the SARS-CoV-2 Omicron Google P. Wang SARS-CoV-2 Omicron variant is to and the PubMed Scopus Google Scholar). The of SARS-CoV-2 variants to and the potential for pandemic to be is to emerging to potential to the efficacy of and the of mutations to of protease and the in vitro activity of nirmatrelvir against the and Omicron variants of Mpro and the structural basis for of in vitro potency against these mutant the for activity against variants mutations in the Mpro and the wildtype Mpro was on J. Hu W. Zhang in vitro of and PubMed Scopus Google Scholar). protease was and the was to each of the variants or The was for of was with of and at of was The was a of and were in 20 with a and the was by at The was by a a by with and a to Mpro and characterization and enzymatic activity of the main protease Mpro of SARS-CoV-2 wildtype and variants was a assay L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google Scholar). The SARS-CoV-2 Mpro assay the activity of SARS-CoV-2 Mpro protease to a with the on a The of the is a on a The assay 20 nM enzyme to nM SARS-CoV-2 Mpro mutant protease was to and enzyme were with the of were from with the to activity from a of enzyme to nM SARS-CoV-2 Mpro mutant protease was to assay in a that in a with and were to for 20 of to to the activity was from the and as activity on activity and enzyme Ki were to the with the enzyme to the to the for each and the to and of SARS-CoV-2 Mpro were in at was with well 20 to and to were at and in was to the and to the for at were a of well and in in for data data were at at of the at a of of for the of the of the at the was by the of the a with The were by and the data as for wildtype SARS-CoV-2 Mpro in with L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). data and for each of the mutant Mpro are in for γ β γ β γ β of in to the in to the in to the in to the in to the in to the as by and for and are the observed and is the as for of the data from in in to the the of the as a for Scopus Google as by and P. and data PubMed Scopus Google and are the observed and in a was at and and with protease and to for of were on the of of in in a and the wildtype Mpro was on J. Hu W. Zhang in vitro of and PubMed Scopus Google Scholar). protease was and the was to each of the variants or The was for of was with of and at of was The was a of and were in 20 with a and the was by at The was by a a by with and a to Mpro and the wildtype Mpro was on J. Hu W. Zhang in vitro of and PubMed Scopus Google Scholar). protease was and the was to each of the variants or The was for of was with of and at of was The was a of and were in 20 with a and the was by at The was by a a by with and a to Mpro and characterization and enzymatic activity of the main protease Mpro of SARS-CoV-2 wildtype and variants was a assay L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google Scholar). The SARS-CoV-2 Mpro assay the activity of SARS-CoV-2 Mpro protease to a with the on a The of the is a on a The assay 20 nM enzyme to nM SARS-CoV-2 Mpro mutant protease was to and enzyme were with the of were from with the to activity from a of enzyme to nM SARS-CoV-2 Mpro mutant protease was to assay in a that in a with and were to for 20 of to to the activity was from the and as activity on activity and enzyme Ki were to the with the enzyme to the to the for each and the to The enzymatic activity of the main protease Mpro of SARS-CoV-2 wildtype and variants was a assay L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google B. L. J. J. characterization of coronavirus protease inhibitor for the potential of 2021; PubMed Scopus Google J. of covalent of coronavirus for the potential therapeutic of PubMed Scopus Google Scholar). The SARS-CoV-2 Mpro assay the activity of SARS-CoV-2 Mpro protease to a with the on a The of the is a on a The assay 20 nM enzyme to nM SARS-CoV-2 Mpro mutant protease was to and enzyme were with the of were from with the to activity from a of enzyme to nM SARS-CoV-2 Mpro mutant protease was to assay in a that in a with and were to for 20 of to to the activity was from the and as activity on activity and enzyme Ki were to the with the enzyme to the to the for each and the to and of SARS-CoV-2 Mpro were in at was with well 20 to and to were at and in was to the and to the for at were a of well and in in for data data were at at of the at a of of for the of the of the at the was by the of the a with The were by and the data as for wildtype SARS-CoV-2 Mpro in with L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). data and for each of the mutant Mpro are in for γ β γ β γ β of in to the in to the in to the in to the in to the in to the as by and for and are the observed and is the as for of the data from in in to the the of the as a for Scopus Google as by and P. and data PubMed Scopus Google and are the observed and in a of SARS-CoV-2 Mpro were in at was with well 20 to and to were at and in was to the and to the for at were a of well and in in for data data were at at of the at a of of for the of the of the at the was by the of the a with The were by and the data as for wildtype SARS-CoV-2 Mpro in with L. B. L. SARS-CoV-2 Mpro inhibitor for the of 2021; PubMed Scopus Google Scholar). data and for each of the mutant Mpro are in is the as for of the data from in Scholar). was at and and with protease and to for of were on the of of in in a was at and and with protease and to for of were on the The and have with the (K90R), (G15S), and (P132H). The and have with the (K90R), (G15S), and (P132H). are of and or of We and for of the and B. and W. B. and J. data W. B. and J. W. B. and J. T. and B. and B. and B. and W. B. T. and and B. J. T. and T. and

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