Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

Jennifer C Milligan,Clovis Basier,Joseph F Curran,Karim Labib,Christelle Soudy,Kang Wei Tan,Mary Wu,John F.X Diffley,George Papageorgiou,Chew Theng Lim,Theresa U Zeisner,Florian Weissmann,Rachel Ulferts,Hema Nagaraj,Dhira Joshi,Berta Canal,Tom Deegan,Rupert Beale,Michael Howell,Ganka Bineva-Todd,Ryo Fujisawa,Nicola O’reilly

doi:10.1042/bcj20210197

Abstract

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Highlights

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, emerged late in 2019 and rapidly spread around the world, developing into the worst health crisis of the 21st century [1,2]
Cleavage of the substrate, which migrates at ∼20 kDa, by nsp5 results in the release of untagged nsp9, which migrates at ∼12 kDa
Two drug candidates targeting nsp5 have entered Phase I clinical trials. Both of them are inhibitors which were initially designed against the nsp5 protease from SARS-CoV-1 [40]

Summary

Introduction

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, emerged late in 2019 and rapidly spread around the world, developing into the worst health crisis of the 21st century [1,2]. As of February 2021, a year after the WHO declared the outbreak a public health emergency of international concern, over 100 million cases have been confirmed, with more than 2.4 million deaths attributed to COVID-19 [3]. New variants of the virus are emerging, and it is unclear how long vaccines will remain effective [5,6]. To combat this pandemic most effectively, antiviral drugs are needed as complements to vaccines

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Conclusion