Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

PICS and TAILS N-Terminomics Characterization of Monkeypox and SARS-CoV-2 protease active sites and identification of novel Anti-viral Neanderthal Gene Products that are inactivated by SARS-CoV-2 Proteases to Circumvent Host Protection to COVID-19 (#45)

Christopher M Overall 1
  1. UBC Centre for Blood Research, Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

Proteases are vital components of viral protein repertoires, and antiviral inhibitor drugs have been game-changers, e.g., HIV AIDS, and hepatitis C. Hence, it is incredibly important to predict the outcome of therapeutic inhibition of the proteases in zoonotic infections and diseases, including COVID-19 and monkeypox. This can only come from understanding the full extent of protein cut sites and proteolysis of host cellular substrates by the viral proteases, yet most studies cherry-pick targets to biochemically screen, and few employ highly validated proteomics analyses targeted to substrate discovery. Further, all investigations of SARS CoV-2 3CLpro “main protease” have focused on the non-prime (P) side to develop anti-viral drugs, neglecting the potential of the prime (P’) side to improve potency. We describe the full cleavage site specificity from P6 to P’6 for SARS-CoV-2 3CLpro and both the cysteine and metalloproteases of monkeypox virus using Proteomic Identification of Cleavage Substrates (PICS) (Nature Biotechnology). For SARS CoV-2 main protease, 3CLpro we also performed 50,000 molecular simulations per peptide in complex with the protease to model 10 of the best fitting sequences to unveil structural determinants of 3CLpro-substrate interaction. Using three proteome peptide libraries (generated by trypsin, GluC and LysargiNase), we determined the preferred cleavage motif of the three proteases from SARS-C0V-2 and monkeypox. Using a total of 816 cleavage sites from CoV-2, we developed soluble and high-efficiency FRET probes to monitor 3CLpro activity. In this analysis, we uncovered a unique target that is a Neanderthal gene product that showed the highest protection from viral infections, yet was found to be a novel SARS-CoV-2 protease substrate. Employing SARDS-CoV-2 infection and transfections of noncleavable vs. cleaved analogues, we show the essential role of this protein and decipher how SARS-CoV-2 defeats its function to lead to worse infection and COVID-19. This analysis has broad implications for extending the chemical envelope of possible chemical routes to improve the treatment of COVID-19, to develop new drug scaffolds prepositioned to adapt to emerging zoonotic Coronavirus spillover to human populations for pandemic preparedness in the future, and, at the same time providing new biological insights into how the virus evades the host immune response and drives replication. Thus, TAILS and PICS are potent terminomics approaches to decipher the pathogenesis of COVID-19, Monkeypox and other emerging viral diseases and to improve anti-viral drug design to treat viral infections now and in the future.