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

Hugo Cesar Ramos de Jesus; Nestor Solis; Yoan Machado; Isabel Pablos; Peter A Bell; Reinhild Kappelhoff; Peter M Grin; Carlos A Sorgi; Georgina S Butler; Christopher M Overall

doi:10.1128/jvi.00049-24

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

J Virol. 2024 May 14:e0004924. doi: 10.1128/jvi.00049-24. Online ahead of print.

Authors

Hugo Cesar Ramos de Jesus^#^{1

2}, Nestor Solis^#^{1

2}, Yoan Machado^{1

2}, Isabel Pablos^{1

2}, Peter A Bell^{1

2}, Reinhild Kappelhoff^{1

2}, Peter M Grin^{1

2}, Carlos A Sorgi^{1

3

4}, Georgina S Butler^{1

2}, Christopher M Overall^{1

2

3

5}

Affiliations

¹ Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.
² Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
³ Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
⁴ Department of Chemistry, Faculty of Philosophy, Sciences and Letters at Ribeirão Preto, University of São Paulo, São Paulo, Brazil.
⁵ Yonsei Frontier Lab, Yonsei University, Seoul, Republic of Korea.

^# Contributed equally.

PMID: 38742901
DOI: 10.1128/jvi.00049-24

Abstract

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^proquenched-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.

Keywords: 3CLpro main protease; COVID-19; Mpro; P' side; PICS; SARS-CoV-2; oxidised methionine; peptide assays; proteomics; structure activity relationship.