Natural Compounds Inhibit SARS-CoV-2 nsp13 Unwinding and ATPase Enzyme Activities

[Image: see text] SARS-CoV-2 infection is still spreading worldwide, and new antiviral therapies are an urgent need to complement the approved vaccine preparations. SARS-CoV-2 nps13 helicase is a validated drug target participating in the viral replication complex and possessing two associated activities: RNA unwinding and 5′-triphosphatase. In the search of SARS-CoV-2 direct antiviral agents, we established biochemical assays for both SARS-CoV-2 nps13-associated enzyme activities and screened both in silico and in vitro a small in-house library of natural compounds. Myricetin, quercetin, kaempferol, and flavanone were found to inhibit the SARS-CoV-2 nps13 unwinding activity at nanomolar concentrations, while licoflavone C was shown to block both SARS-CoV-2 nps13 activities at micromolar concentrations. Mode of action studies showed that all compounds are nsp13 noncompetitive inhibitors versus ATP, while computational studies suggested that they can bind both nucleotide and 5′-RNA nsp13 binding sites, with licoflavone C showing a unique pattern of interaction with nsp13 amino acid residues. Overall, we report for the first time natural flavonoids as selective inhibitors of SARS-CoV-2 nps13 helicase with low micromolar activity.