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

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. [Display omitted] •We developed a highly sensitive cell-based Nanoluciferase assay to screen for inhibitors of SARS-CoV-2 main protease.•We provide evidence that the assay is scalable to the 384-well format and robust (z’ factor of 0.8).•The false positive rate is minimized: gain of signal assay, catalytically inactive protease control.•The assay can be adapted to other coronaviruses and viruses which rely on a viral protease.•We report the identification of 2 lead compounds that inhibit nsp5 activity and SARS-CoV-2 replication in cell culture.