Repurposing drug screen for the identification of helicase inhibitors from viruses of pandemic concern.

Several families of viruses encode helicase enzymes with essential roles in viral genome replication and transcription, including several RNA virus families of pandemic concern, such as coronaviruses and flaviviruses. Viral helicases are widely considered to be promising antiviral targets with some DNA virus helicase inhibitors already approved for clinical use. Here, we demonstrate that the SARS-CoV-2 viral helicase, nsp13, is essential for viral replication. Using a previously developed in vitro helicase dsRNA unwinding assay, we screened 208 repurposed small molecules for nsp13 inhibition. Nine of these small molecules had a half-maximal inhibitory concentration (IC50) <10 µM. These molecules were tested for in vitro inhibition of nsp13 ATPase activity as well as for inhibition of Yellow Fever Virus (YFV) NS3 helicase (NS3h) activity. FPA-124, a selective inhibitor of AKT kinase, was found to inhibit the unwinding activity of both SARS-CoV-2 nsp13 and YFV NS3h, though it only inhibited ATPase activity of nsp13. Molecular ligand docking confirmed that FPA-124 likely binds within the ATP binding pocket of nsp13, but not YFV NS3h. Unfortunately, FPA-124 had a low selective index in cultured lung cells, with an IC50 against SARS-CoV-2 near the half-maximal cytotoxic concentration (CC50) in cells. Overall, we developed several high-throughput assays for antiviral drug screening against viral helicases and identified several lead scaffolds that may provide much needed tools to address future pandemics and endemic diseases with few treatment options.