Oral Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2 (#5)

Theresa Klemm 1 , Gregor Ebert 1 , Dale J Calleja 1 , Cody C Allison 1 , Lachlan W Richardson 1 , Jonathan P Bernardini 1 2 , Bernadine G C Lu 1 , Nathan W Kuchel 1 , Christoph Grohmann 1 , Yuri Shibata 1 , Zhong Yan Gan 1 , James P Cooney 1 , Marcel Doerflinger 1 , Amanda E Au 1 , Timothy R Blackmore 1 , Gerbrand J Heden van Noort 3 , Paul P Geurink 3 , Huib Ovaa 3 , Janet Newman 4 , Alan Tunnicliffe-Riboldi 5 , Peter E Czabotar 1 , Jeffrey P Mitchell 1 , Rebecca Feltham 1 , Bernhard C Lechtenberg 1 , Kym N Lowes 1 , Grant Dewson 1 , Marc Pellegrini 1 , Guillaume Lessene 1 6 , David Komander 1
  1. WEHI, Melbourne/ Parkville, VIC, Australia
  2. Department of Biochemistry and Molecular Biology,, Michael Smith Laboratories University of British Columbia, Vancouver, BC, Canada
  3. Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, Netherlands
  4. Commonwealth Scientific and Industrial Research Organisation (CSIRO), Biomedical Program, Parkville, VIC, Australia
  5. Australian Synchrotron, ANSTO, Clayton, VIC, Australia
  6. Pharmacology and Therapeutics Department, University of Melbourne, Melbourne, VIC, Australia

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications. Drugs that target SARS-CoV-2 PLpro (SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19 reducing viral load and reinstating innate immune responses.

In our study we characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves ISG15 modifications with high activity, as well as, Lys-48-linked polyubiquitin with lower activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin‐binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We exploit two repurposing approaches to target SARS2 PLpro: A high throughput screen against SARS2 PLpro using a library of 3,727 unique approved drugs and clinical compounds identified no compounds that inhibited PLpro consistently or that could be validated in counter screens. More promisingly, non-covalent small molecules SARS PLpro inhibitors, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.