Proteomic identification of SARS-CoV-2 3CL<sup>pro</sup> P6—P6' active site specificity to inform optimization of superior quenched fluorescent peptide substrates

Proteomic identification of SARS-CoV-2 3CL^pro P6—P6' active site specificity to inform optimization of superior quenched fluorescent peptide substrates

Proteomic identification of SARS-CoV-2 3CL^pro P6—P6' active site specificity to inform optimization of superior quenched fluorescent peptide substrates (#59)

Chris Overall ¹

UBC Centre for Blood Research, Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

SARS-CoV-2 chymotrypsin-like (3CL^pro) main protease is crucial for viral protein maturation and is a validated therapeutic target to block SARS-CoV-2 replication. 3CL^pro inhibitor development focuses mainly on the catalytic non-prime (P) side, whereas the contribution of the prime side (P') remains under-described. We determined the P6 – P6' specificity for SARS-CoV-2 3CL^pro from analysis of >800 cleavage sites identified using “Proteomic Identification of Cleavage site Specificity” (PICS). In addition to specificity validation using synthetic peptides and positional libraries, molecular docking simulations unveiled key structure-activity relationships impacting 3CL^pro-substrate interactions. The S3' subsite displays marked flexibility with an unreported P3'-His specificity that forms backbone h-bonds to Thr²⁵centrally located in a striking ‘threonine triad’ between Thr²⁴ – Thr²⁶ in the 3CL^pro P'-binding domain I. Rather than cleavage at P1-glutamine, cleavage also occurs after non-canonical Met and His residues. Essential h-bonds between the N-terminal Ser¹ of protomer B in the 3CL^pro dimer and P1-His form, but not for Met. Nonetheless, MAP4K5 is cleaved after Met⁴⁵⁶. We show the h-bonding potential of oxidized P1-Met with Ser¹ and the cleavage of corresponding synthetic peptides, which is relevant as intracellular reactive oxygen species are elevated in infection. Our analyses detailing previously overlooked P'-side and novel P-side structural determinants were explored to design two new highly soluble peptide substrates for 3CL^pro exhibiting 15x improved sensitivity over current peptide assays.

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Overall, C

Professor Christopher Overall is a Tier 1 Canada Research Chair in Protease Proteomics and Systems Biology and a Senior Fellow of the Freiburg Institute of Advanced Studies, Albert-Ludwigs Universität Freiburg, Germany (2010–2013), where he is now an Honorary Professor. He was inducted as a fellow into the Royal Society of Canada in 2018. Chris is best known for developing proteomic methodology for discovering protease substrates in vivo, thereby establishing the field of degradomics. By generating clinically relevant insights into how proteases dampen disease-fighting defence systems involved in chemokine and complement regulation of macrophages and neutrophils in inflammation, viral host resistance, and immunodeficiency degradomics has revealed a new layer of complexity in the hierarchy of cell and immune signalling regulation, revolutionizing our understanding of protease function and drug targeting. These insights led to developing a new class of molecular correctors to treat MALT-1 protease deficiency and NF-kB activation in immunodeficiency disease. During the pandemic, Chris pivoted his laboratory to the investigation of SARS-CoV-2 proteases, revealing their drastic impact on cleaving hundreds of host cell proteins in critical pathways that collectively promote the cellular takeover by the virus. Dr. Overall completed his undergraduate and Master’s degrees at the University of Adelaide, South Australia; his Ph.D. in Biochemistry at the University of Toronto, Canada; and was an MRC Centennial Fellow in his postdoctoral work with Dr. Michael Smith, Nobel Laureate, UBC. He was a Senior Scientist at British Biotech, Oxford, and at the Protease Platform, Centre for Proteomic Drug Discovery, Novartis, Basel. His papers are influential, with an h-index of 102, and his advances in proteomics have been recognized by the Canadian National Proteomics Network Tony Pawson Award (2014); the Proteomass Scientific Society Award (2017); the 2018 international HUPO Discovery Award in Proteomics Sciences; and the 2022 Helmut Holzer Award. He is an Associate Editor of the Journal of Proteome Research and Chairs the HUPO Chromosome-centric Human Proteome Project (C-HPP).

Proteomic identification of SARS-CoV-2 3CLpro P6—P6' active site specificity to inform optimization of superior quenched fluorescent peptide substrates (#59)

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Proteomic identification of SARS-CoV-2 3CL^pro P6—P6' active site specificity to inform optimization of superior quenched fluorescent peptide substrates (#59)