{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T03:03:30Z","timestamp":1787022210866,"version":"build-2736575974"},"update-to":[{"DOI":"10.1371\/journal.ppat.1011592","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T00:00:00Z","timestamp":1694563200000}}],"reference-count":51,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2023,8,31]],"date-time":"2023-08-31T00:00:00Z","timestamp":1693440000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["1R15GM129766-01"],"award-info":[{"award-number":["1R15GM129766-01"]}]},{"name":"NIH","award":["AI158775"],"award-info":[{"award-number":["AI158775"]}]},{"name":"NSF","award":["2200138"],"award-info":[{"award-number":["2200138"]}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>\n                    The SARS-CoV-2 main protease (M\n                    <jats:sup>pro<\/jats:sup>\n                    ) is a major therapeutic target. The M\n                    <jats:sup>pro<\/jats:sup>\n                    inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As M\n                    <jats:sup>pro<\/jats:sup>\n                    inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for M\n                    <jats:sup>pro<\/jats:sup>\n                    activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of M\n                    <jats:sup>pro<\/jats:sup>\n                    E166R, and M\n                    <jats:sup>pro<\/jats:sup>\n                    E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of M\n                    <jats:sup>pro<\/jats:sup>\n                    that may arise as M\n                    <jats:sup>pro<\/jats:sup>\n                    antivirals become more widely used.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1011592","type":"journal-article","created":{"date-parts":[[2023,8,31]],"date-time":"2023-08-31T13:43:12Z","timestamp":1693489392000},"page":"e1011592","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":24,"title":["A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations"],"prefix":"10.1371","volume":"19","author":[{"given":"Jin","family":"Ou","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric M.","family":"Lewandowski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanmei","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Austin A.","family":"Lipinski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Aljasser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariliz","family":"Colon-Ascanio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan T.","family":"Morgan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lian M. C.","family":"Jacobs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiujun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa J.","family":"Bikowitz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul R.","family":"Langlais","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haozhou","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9196-3280","authenticated-orcid":true,"given":"John S.","family":"Choy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,8,31]]},"reference":[{"issue":"5","key":"ppat.1011592.ref001","article-title":"Antiviral Drug Discovery for the Treatment of COVID-19 Infections","volume":"14","author":"TI Ng","year":"2022","journal-title":"Viruses"},{"issue":"7811","key":"ppat.1011592.ref002","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1038\/s41586-020-2223-y","article-title":"Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors","volume":"582","author":"Z Jin","year":"2020","journal-title":"Nature"},{"issue":"1","key":"ppat.1011592.ref003","doi-asserted-by":"crossref","first-page":"e202100576","DOI":"10.1002\/cmdc.202100576","article-title":"A Patent Review on SARS Coronavirus Main Protease (3CL(pro)) Inhibitors","volume":"17","author":"CSB Chia","year":"2022","journal-title":"ChemMedChem"},{"issue":"9","key":"ppat.1011592.ref004","article-title":"Discovery of M Protease Inhibitors Encoded by SARS-CoV-2","volume":"64","author":"HC Hung","year":"2020","journal-title":"Antimicrob Agents Chemother"},{"issue":"12","key":"ppat.1011592.ref005","doi-asserted-by":"crossref","first-page":"e0243697","DOI":"10.1371\/journal.pone.0243697","article-title":"COVID-19 and human-nature relationships: Vermonters\u2019 activities in nature and associated nonmaterial values during the pandemic","volume":"15","author":"JW Morse","year":"2020","journal-title":"PLoS One"},{"key":"ppat.1011592.ref006","unstructured":"FDA. Fact sheet for healthcare providers: Emergency use authorization for Paxlovid 2021. Available from: https:\/\/www.fda.gov\/media\/155050\/download."},{"issue":"6575","key":"ppat.1011592.ref007","doi-asserted-by":"crossref","first-page":"1586","DOI":"10.1126\/science.abl4784","article-title":"An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19","volume":"374","author":"DR Owen","year":"2021","journal-title":"Science"},{"issue":"15","key":"ppat.1011592.ref008","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.1056\/NEJMoa2118542","article-title":"Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19","volume":"386","author":"J Hammond","year":"2022","journal-title":"N Engl J Med"},{"issue":"1","key":"ppat.1011592.ref009","doi-asserted-by":"crossref","first-page":"6055","DOI":"10.1038\/s41467-021-26239-2","article-title":"Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19","volume":"12","author":"B Boras","year":"2021","journal-title":"Nat Commun"},{"issue":"1","key":"ppat.1011592.ref010","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/d41573-021-00202-8","article-title":"A tale of two antiviral targets\u2014and the COVID-19 drugs that bind them","volume":"21","author":"M. Cully","year":"2022","journal-title":"Nat Rev Drug Discov."},{"issue":"10","key":"ppat.1011592.ref011","doi-asserted-by":"crossref","first-page":"e0069722","DOI":"10.1128\/aac.00697-22","article-title":"A Randomized Phase 2\/3 Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part","volume":"66","author":"H Mukae","year":"2022","journal-title":"Antimicrob Agents Chemother"},{"key":"ppat.1011592.ref012","volume-title":"Information for laboratories about Coronavirus (COVID-19)","author":"CDC","year":"2020"},{"issue":"5","key":"ppat.1011592.ref013","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1021\/acsptsci.0c00108","article-title":"Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-Throughput Screening","volume":"3","author":"W Zhu","year":"2020","journal-title":"ACS Pharmacol Transl Sci"},{"issue":"1","key":"ppat.1011592.ref014","doi-asserted-by":"crossref","first-page":"e0281522","DOI":"10.1128\/mbio.02815-22","article-title":"The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir","volume":"14","author":"D Jochmans","year":"2023","journal-title":"mBio"},{"key":"ppat.1011592.ref015","article-title":"Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro","author":"Y Zhou","year":"2022","journal-title":"bioRxiv"},{"issue":"10","key":"ppat.1011592.ref016","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.1016\/j.chom.2022.08.003","article-title":"Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites","volume":"30","author":"S Iketani","year":"2022","journal-title":"Cell Host Microbe"},{"issue":"7944","key":"ppat.1011592.ref017","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1038\/s41586-022-05514-2","article-title":"Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir","volume":"613","author":"S Iketani","year":"2023","journal-title":"Nature"},{"issue":"3","key":"ppat.1011592.ref018","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/s41579-020-00468-6","article-title":"Coronavirus biology and replication: implications for SARS-CoV-2","volume":"19","author":"P V\u2019Kovski","year":"2021","journal-title":"Nat Rev Microbiol"},{"issue":"12","key":"ppat.1011592.ref019","doi-asserted-by":"crossref","first-page":"e28479","DOI":"10.1371\/journal.pone.0028479","article-title":"Yeast based small molecule screen for inhibitors of SARS-CoV","volume":"6","author":"M Frieman","year":"2011","journal-title":"PLoS One"},{"issue":"1","key":"ppat.1011592.ref020","doi-asserted-by":"crossref","first-page":"3202","DOI":"10.1038\/s41467-020-16954-7","article-title":"Structural plasticity of SARS-CoV-2 3CL M(pro) active site cavity revealed by room temperature X-ray crystallography","volume":"11","author":"DW Kneller","year":"2020","journal-title":"Nat Commun"},{"issue":"50","key":"ppat.1011592.ref021","doi-asserted-by":"crossref","DOI":"10.1126\/sciadv.abe0751","article-title":"Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L","volume":"6","author":"MD Sacco","year":"2020","journal-title":"Sci Adv"},{"issue":"6497","key":"ppat.1011592.ref022","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1126\/science.abb4489","article-title":"Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease","volume":"368","author":"W Dai","year":"2020","journal-title":"Science"},{"key":"ppat.1011592.ref023","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1099\/0022-1317-83-3-595","article-title":"Conservation of substrate specificities among coronavirus main proteases","volume":"83","author":"A Hegyi","year":"2002","journal-title":"J Gen Virol"},{"issue":"1","key":"ppat.1011592.ref024","doi-asserted-by":"crossref","first-page":"3556","DOI":"10.1038\/s41467-022-31210-w","article-title":"Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance","volume":"13","author":"AM Shaqra","year":"2022","journal-title":"Nat Commun"},{"issue":"6","key":"ppat.1011592.ref025","doi-asserted-by":"crossref","first-page":"3238","DOI":"10.1021\/acs.chemrev.0c00648","article-title":"Drug Design Strategies to Avoid Resistance in Direct-Acting Antivirals and Beyond","volume":"121","author":"AN Matthew","year":"2021","journal-title":"Chem Rev"},{"issue":"4","key":"ppat.1011592.ref026","doi-asserted-by":"crossref","first-page":"1965","DOI":"10.1002\/med.21783","article-title":"What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design","volume":"41","author":"M Xiong","year":"2021","journal-title":"Med Res Rev"},{"issue":"9","key":"ppat.1011592.ref027","doi-asserted-by":"crossref","first-page":"2591","DOI":"10.1021\/acsinfecdis.1c00237","article-title":"Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease","volume":"7","author":"EA MacDonald","year":"2021","journal-title":"ACS Infect Dis"},{"issue":"5","key":"ppat.1011592.ref028","doi-asserted-by":"crossref","first-page":"2515","DOI":"10.1128\/JVI.02114-07","article-title":"Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design","volume":"82","author":"X Xue","year":"2008","journal-title":"J Virol"},{"issue":"6","key":"ppat.1011592.ref029","doi-asserted-by":"crossref","first-page":"101972","DOI":"10.1016\/j.jbc.2022.101972","article-title":"Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants","volume":"298","author":"SE Greasley","year":"2022","journal-title":"J Biol Chem"},{"issue":"5","key":"ppat.1011592.ref030","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1038\/s41422-022-00640-y","article-title":"The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition","volume":"32","author":"MD Sacco","year":"2022","journal-title":"Cell Res"},{"key":"ppat.1011592.ref031","doi-asserted-by":"crossref","first-page":"128629","DOI":"10.1016\/j.bmcl.2022.128629","article-title":"Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir","volume":"62","author":"S Ullrich","year":"2022","journal-title":"Bioorg Med Chem Lett"},{"issue":"3","key":"ppat.1011592.ref032","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1038\/s41422-022-00618-w","article-title":"SARS-CoV-2 Omicron variant is highly sensitive to molnupiravir, nirmatrelvir, and the combination","volume":"32","author":"P Li","year":"2022","journal-title":"Cell Res"},{"issue":"4","key":"ppat.1011592.ref033","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1016\/j.jmb.2011.03.038","article-title":"Accessory mutations maintain stability in drug-resistant HIV-1 protease","volume":"410","author":"MW Chang","year":"2011","journal-title":"J Mol Biol"},{"key":"ppat.1011592.ref034","volume-title":"Antiretroviral Resistance in Clinical Practice","author":"A. De Luca","year":"2006"},{"key":"ppat.1011592.ref035","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.copbio.2021.10.014","article-title":"Exploring the biological function of efflux pumps for the development of superior industrial yeasts","volume":"74","author":"I Sa-Correia","year":"2022","journal-title":"Curr Opin Biotechnol"},{"issue":"12","key":"ppat.1011592.ref036","doi-asserted-by":"crossref","first-page":"3695","DOI":"10.1128\/AAC.46.12.3695-3705.2002","article-title":"Drug susceptibilities of yeast cells are affected by membrane lipid composition","volume":"46","author":"K Mukhopadhyay","year":"2002","journal-title":"Antimicrob Agents Chemother"},{"issue":"1","key":"ppat.1011592.ref037","doi-asserted-by":"crossref","first-page":"5553","DOI":"10.1038\/s41467-021-25796-w","article-title":"Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential","volume":"12","author":"B Meyer","year":"2021","journal-title":"Nat Commun"},{"issue":"4","key":"ppat.1011592.ref038","doi-asserted-by":"crossref","first-page":"109892","DOI":"10.1016\/j.celrep.2021.109892","article-title":"Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL(pro) substrate degradome","volume":"37","author":"I Pablos","year":"2021","journal-title":"Cell Rep"},{"issue":"6","key":"ppat.1011592.ref039","doi-asserted-by":"crossref","first-page":"e0108721","DOI":"10.1128\/mSystems.01087-21","article-title":"A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease","volume":"6","author":"H Alalam","year":"2021","journal-title":"mSystems"},{"key":"ppat.1011592.ref040","first-page":"11","article-title":"Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms","author":"JM Flynn","year":"2022","journal-title":"Elife"},{"key":"ppat.1011592.ref041","article-title":"Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes","author":"AK Padhi","year":"2022","journal-title":"bioRxiv"},{"key":"ppat.1011592.ref042","article-title":"Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir","author":"Y Hu","year":"2022","journal-title":"bioRxiv"},{"issue":"2","key":"ppat.1011592.ref043","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1534\/genetics.118.301727","article-title":"Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in Saccharomyces cerevisiae","volume":"211","author":"SB Shah","year":"2019","journal-title":"Genetics"},{"issue":"7","key":"ppat.1011592.ref044","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"J Schindelin","year":"2012","journal-title":"Nat Methods"},{"issue":"1","key":"ppat.1011592.ref045","doi-asserted-by":"crossref","first-page":"E67","DOI":"10.1152\/ajpendo.00057.2020","article-title":"Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction","volume":"319","author":"AL Pendleton","year":"2020","journal-title":"Am J Physiol Endocrinol Metab"},{"issue":"7","key":"ppat.1011592.ref046","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1074\/mcp.RA119.001450","article-title":"Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes","volume":"18","author":"SS Parker","year":"2019","journal-title":"Mol Cell Proteomics"},{"issue":"9","key":"ppat.1011592.ref047","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1038\/nmeth.3901","article-title":"The Perseus computational platform for comprehensive analysis of (prote)omics data","volume":"13","author":"S Tyanova","year":"2016","journal-title":"Nat Methods"},{"key":"ppat.1011592.ref048","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1007\/978-1-4939-7493-1_7","article-title":"Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research","volume":"1711","author":"S Tyanova","year":"2018","journal-title":"Methods Mol Biol"},{"issue":"1","key":"ppat.1011592.ref049","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1038\/nprot.2008.211","article-title":"Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources","volume":"4","author":"W Huang da","year":"2009","journal-title":"Nat Protoc"},{"key":"ppat.1011592.ref050","article-title":"Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture","author":"Y Hu","year":"2020","journal-title":"bioRxiv"},{"issue":"8","key":"ppat.1011592.ref051","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1038\/s41422-020-0356-z","article-title":"Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease","volume":"30","author":"C Ma","year":"2020","journal-title":"Cell Res"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1011592","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T00:00:00Z","timestamp":1694563200000}}],"container-title":["PLOS Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1011592","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T13:37:53Z","timestamp":1694612273000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1011592"}},"subtitle":[],"editor":[{"given":"Wenshe","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2023,8,31]]},"references-count":51,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2023,8,31]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.ppat.1011592","relation":{},"ISSN":["1553-7374"],"issn-type":[{"value":"1553-7374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,31]]}}}