Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

  1. Maria Kuzikov
  2. Elisa Costanzi
  3. Jeanette Reinshagen
  4. Francesca Esposito
  5. Laura Vangeel
  6. Markus Wolf
  7. Bernhard Ellinger
  8. Carsten Claussen
  9. Gerd Geisslinger
  10. Angela Corona
  11. Daniela Iaconis
  12. Carmine Talarico
  13. Candida Manelfi
  14. Rolando Cannalire
  15. Giulia Rossetti
  16. Jonas Gossen
  17. Simone Albani
  18. Francesco Musiani
  19. Katja Herzog
  20. Yang Ye
  21. Barbara Giabbai
  22. Nicola Demitri
  23. Dirk Jochmans
  24. Steven De Jonghe
  25. Jasper Rymenants
  26. Vincenzo Summa
  27. Enzo Tramontano
  28. Andrea R. Beccari
  29. Pieter Leyssen
  30. Paola Storici
  31. Johan Neyts
  32. Philip Gribbon
  33. Andrea Zaliani
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Abstract

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 μM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

Graphical abstract

<fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><label>Abstract Figure.</label><caption>

Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray.

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