{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T19:34:42Z","timestamp":1793388882949,"version":"4.5.0"},"reference-count":32,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2024,6,13]],"date-time":"2024-06-13T00:00:00Z","timestamp":1718236800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2024,6,13]],"date-time":"2024-06-13T00:00:00Z","timestamp":1718236800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Canadian Government | Canadian Institutes of Health Research","award":["148408"],"award-info":[{"award-number":["148408"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2024,6,13]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:sec>\n            <jats:title\/>\n            <jats:p>\n              SARS-CoV-2 3C-like main protease (3CL\n              <jats:sup>pro<\/jats:sup>\n              ) is essential for protein excision from the viral polyprotein. 3CL\n              <jats:sup>pro<\/jats:sup>\n              inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6\u2013P6' specificity for 3CL\n              <jats:sup>pro<\/jats:sup>\n              from &gt;800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg\/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CL\n              <jats:sup>pro<\/jats:sup>\n              dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-Met\n              <jats:sub>OX<\/jats:sub>\n              forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3\u2032-His that displays stabilizing backbone H-bonds with Thr25 lying central in a \u201c\u2018threonine trio\u201d (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CL\n              <jats:sup>pro<\/jats:sup>\n              -substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CL\n              <jats:sup>pro<\/jats:sup>\n              quenched-fluorescent peptide substrates for improved FRET monitoring of 3CL\n              <jats:sup>pro<\/jats:sup>\n              activity with 15\u00d7 improved sensitivity over current assays.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                From global proteomics identification of &gt;800 cleavage sites, we characterized the P6\u2013P6' active site specificity of SARS-CoV-2 3CL\n                <jats:sup>pro<\/jats:sup>\n                using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3\u00d7 faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-Met\n                <jats:sub>OX<\/jats:sub>\n                ). We unveiled unexpected amino acid cooperativity at P1-Met with P3\u2032-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CL\n                <jats:sup>pro<\/jats:sup>\n                . From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with &gt;15\u00d7 improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/jvi.00049-24","type":"journal-article","created":{"date-parts":[[2024,5,14]],"date-time":"2024-05-14T13:01:26Z","timestamp":1715691686000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":11,"title":["Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CL\n            <sup>pro<\/sup>\n            main protease (Mpro) from proteomic identification of P6\u2014P6' active site specificity"],"prefix":"10.1128","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8223-094X","authenticated-orcid":false,"given":"Hugo","family":"Cesar Ramos de Jesus","sequence":"first","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8208-0352","authenticated-orcid":false,"given":"Nestor","family":"Solis","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8251-3744","authenticated-orcid":false,"given":"Yoan","family":"Machado","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8052-3853","authenticated-orcid":false,"given":"Isabel","family":"Pablos","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6143-5444","authenticated-orcid":false,"given":"Peter A.","family":"Bell","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9810-7751","authenticated-orcid":false,"given":"Reinhild","family":"Kappelhoff","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9504-9664","authenticated-orcid":false,"given":"Peter M.","family":"Grin","sequence":"additional","affiliation":[{"name":"Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada"},{"name":"Department of 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