The RdRp Thumb-1 Pocket is a Conserved Target for Broad-Spectrum Antiviral Development

RNA viruses cause human diseases ranging from mild colds to deadly pandemics. Direct-acting, broad-spectrum, non-nucleoside antivirals have been characterized as impossible to develop because allosteric binding sites are poorly conserved. The HCV NS5B RNA-dependent RNA polymerase (RdRp) Thumb-1 allosteric site and its interaction with the HCV NS5B {Lambda}1-loop governs an essential conformational change required for polymerase initiation. The only approved NS5B Thumb-1 inhibitor, beclabuvir, has been shown to be inactive against a broad panel of non-HCV viruses, including poliovirus, rhinovirus, coronavirus, coxsackievirus, influenzavirus, and HIV. A conserved, homologous allosteric site on RdRp that spans multiple viral families has not been reported. Here, we report GALILEO's discovery that the Thumb-1 pocket, its associated {Lambda}1-loop and their interaction are conserved across RNA viral families for the first time. The discovery is validated through comparative structural analysis of Protein Data Bank (PDB) deposited viral polymerases utilizing a method that allows independent, public validation by any researcher. We further demonstrate that beclabuvir's dependence on its indole C6 carbonyl to interact with the HCV-specific residue R503 restricts its activity to HCV. We validate the target discovery with MDL-001, which does not contain a C6 carbonyl substituent. MDL-001 directly blocks viral RNA synthesis in isolated replication complexes and selects for the canonical Thumb-1 resistance mutation P495S in HCV NS5B. MDL-001 demonstrates broad-spectrum in vitro inhibition of both HCV and SARS-CoV-2. Preclinical proof of concept and development of MDL-001 across HCV, HBV, HDV, influenza, SARS-CoV-2, and RSV have been previously reported. These findings establish RdRp Thumb-1 as a conserved allosteric pocket and a druggable target for broad-spectrum direct-acting antiviral development.