Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Deep Mutational Scanning of SARS-CoV-2 Proteases (#155)

Xinyu Wu 1 2 , Margareta Go 1 , Matthew Call 1 2 , Melissa Call 1 2
  1. Walter and Eliza Institite of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

SARS-CoV-2 is now moving from being the causative agent of the pandemic to becoming an ever present part of life. Nevertheless, SARS-CoV-2 will be a leading cause of death in developed nations for the foreseeable future and therapies to ameliorate this burden should be developed. Mpro and PLpro are proteases that cleave viral polyproteins into functional proteins and are required for viral replication. As such they are the subject of major drug-discovery programs around the world and at WEHI1,2. Pfizer successfully launched the drug Paxlovid, which contains an Mpro inhibitor to stop virus replication3,4. However, Mpro variants that are insensitive to Paxlovid have already been reported in laboratory and the wide distribution of Paxlovid would provide selective pressure for such drug-resistant mutants prevail5. Additional drugs should be developed before existing treatments fail.

 

To complement drug-discovery efforts at WEHI for Mpro and papain-like protease (PLpro) inhibitors, we have developed methods to perform deep mutational scanning (DMS) on these targets.  Deep mutational scanning is an approach that functionally assesses the impact of every possible single-site substitution in a protein and provides a wealth of data about how a protein functions and which mutations might cause resistance to lead compounds while they are in development6. 

 

To perform DMS on Mpro we have established a survival assay that takes advantage of the fact that Mpro is toxic to cells and this is dependent on Mpro’s catalytic activity. For PLpro we have established a FRET biosensor that is cleaved by PLpro and measured by flow cytometry. We constructed DMS libraries for Mpro and PLpro with cutting-edge library construction methods7. The deep mutational scanning data aligns with previously published reports on these proteases and hints several interesting drug escape mutants and other regions of the protease that might be targeted by drug discovery efforts.  

  1. Amin, S. A., Banerjee, S., Ghosh, K., Gayen, S. & Jha, T. Protease targeted COVID-19 drug discovery and its challenges: Insight into viral main protease (Mpro) and papain-like protease (PLpro) inhibitors. Bioorg. Med. Chem. 29, 115860 (2021).
  2. Jin, Z. et al. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature 582, 289–293 (2020).
  3. Li, J. et al. Structural basis of main proteases of coronavirus bound to drug candidate PF-07321332. bioRxiv (2021).
  4. Owen, D. R. et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science (80-. ). eabl4784 (2021).
  5. Jochmans, D. et al. The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir. bioRxiv (2022).
  6. Araya, C. L. & Fowler, D. M. Deep mutational scanning: assessing protein function on a massive scale. Trends Biotechnol. 29, 435–442 (2011).
  7. Melnikov, A., Rogov, P., Wang, L., Gnirke, A. & Mikkelsen, T. S. Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes. Nucleic Acids Res. 42, e112–e112 (2014).