Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CL pro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

Autor:	Cesar Ramos de Jesus H; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Solis N; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Machado Y; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Pablos I; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Bell PA; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Kappelhoff R; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Grin PM; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Sorgi CA; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.; Department of Chemistry, Faculty of Philosophy, Sciences and Letters at Ribeirão Preto, University of São Paulo, São Paulo, Brazil., Butler GS; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada., Overall CM; Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.; Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.; Yonsei Frontier Lab, Yonsei University, Seoul, Republic of Korea.
Jazyk:	angličtina
Zdroj:	Journal of virology [J Virol] 2024 Jun 13; Vol. 98 (6), pp. e0004924. Date of Electronic Publication: 2024 May 14.
DOI:	10.1128/jvi.00049-24
Abstrakt:	SARS-CoV-2 3C-like main protease (3CL pro ) is essential for protein excision from the viral polyprotein. 3CL pro 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-P6' specificity for 3CL pro from >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 pro 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 OX 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'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CL pro -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 pro quenched-fluorescent peptide substrates for improved FRET monitoring of 3CL pro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CL pro 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× 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 OX ). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-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 pro . From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs. Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
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