Identification and characterization of a non-nucleotide RdRp allosteric inhibitor with pan-coronavirus protection

The viral RNA-dependent RNA polymerase (RdRp) is a conserved and compelling target for pan-coronavirus antiviral development. To date, all US Food and Drug Administration (FDA)-approved RdRp inhibitors are nucleotide inhibitors (NIs), which are prone to drug resistance and show limited clinical efficacy. Developing alternative non-nucleotide inhibitors (NNIs) with high target specificity, structural diversity, and metabolic stability could yield more effective antivirals. Here, we identify an allosteric RdRp NNI, BAY-850, via computational screening. BAY-850 exhibits potent, broad-spectrum antiviral activity against multiple SARS-CoV-2 variants and other human coronaviruses. Mechanistic studies show that BAY-850 binds directly to a previously unrecognized allosteric site on nsp12, thereby fine-tuning the catalytic motif F loop out-conformation the orthosteric site. Importantly, therapeutic administration of BAY-850 significantly reduces viral loads in the nasal turbinates and lungs of SARS-CoV-2-infected K18-hACE2 mice. These findings validate a novel allosteric site in RdRp and support BAY-850 as a start-point for broad-spectrum anti-coronavirus development.