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

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 micromolar 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.

Nonlinear Regression Modelling: A Primer with Applications and Caveats

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A community effort to discover small molecule SARS-CoV-2 inhibitors

“…The Pasteur Institute in Paris had previously set up a cell-based Nsp5 protease assay, 73 in which cleavage of a reporter Rev-Nluc protein by Nsp5 decreases the luminescence signal. In the presence of an inhibitor, the luminescence signal is restored (see Supporting Information Section 4.3).…”