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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 EC 50 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. [ABSTRACT FROM AUTHOR]

In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals. • The genomic sequence of 2019-nCoV indicates that the virus clusters with betacoronaviruses of lineage b. • 2019-nCoV S-protein sequence has a specific furin-like cleavage site absent in lineage b CoV including SARS-CoV sequences. • The furin-like cleavage site in the S-protein of 2019-nCoV may have implications for the viral life cycle and pathogenicity. • Campaigns to develop anti-2019-nCoV therapeutics should include the evaluation of furin inhibitors. [ABSTRACT FROM AUTHOR]