A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors
Kuang‐Yu Chen, Tim Krischuns, Laura Ortega Varga, Emna Harigua‐Souiai, Sylvain Paisant, Agnès Zettor, Jeanne Chiaravalli, Adrien Delpal, David Courtney, Amornrat O’Brien, Susan C. Baker, Étienne Decroly, Catherine Isel, Fabrice Agou, Yves Jacob, Arnaud Blondel, Nadia Naffakh
Abstract
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4–8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.