A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Chen, K.-Y.; Krischuns, T.; Ortega Varga, L.; Harigua-Souiai, E.; Paisant, S.; Zettor, A.; Chiaravalli, J.; Courtney, D.; O'Brien, A.; Baker, S.; Isel, C.; Agou, F.; Jacob, Y.; Blondel, A.; Naffakh, N.

doi:10.1101/2021.12.18.473303

bioRxiv · 10.1101/2021.12.18.473303

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Chen, K.-Y.·Krischuns, T.·Ortega Varga, L.·Harigua-Souiai, E.·Paisant, S.·Zettor, A.·Chiaravalli, J.·Courtney, D.·O'Brien, A.·Baker, S.·Isel, C.·Agou, F.·Jacob, Y.·Blondel, A.·Naffakh, N.

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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 high-throughput 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 four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 {micro}M range. The here described 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.

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Chen, K.-Y., Krischuns, T., Ortega Varga, L., Harigua-Souiai, E., Paisant, S., Zettor, A., Chiaravalli, J., Courtney, D., O'Brien, A., Baker, S., Isel, C., Agou, F., Jacob, Y., Blondel, A., Naffakh, N.. 2021-12-21. A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors. https://doi.org/10.1101/2021.12.18.473303

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