High-throughput screening identifies non-nucleoside inhibitors of the SARS-CoV-2 polymerase with novel mechanisms.

The RNA-dependent RNA polymerase (RdRp) of coronaviruses, comprising highly conserved non-structural proteins, is a critical player in the viral lifecycle and represents a promising target for developing pan-coronavirus antivirals. Despite substantial efforts to identify RdRp inhibitors through drug repurposing and novel compound discovery campaigns, potent in vitro non-nucleoside inhibitors remain elusive. In this study, we detail the development of a robust PicoGreen assay, which facilitated the screening of AbbVie's extensive chemical library, encompassing over 900,000 small molecules, against the SARS-CoV-2 RdRp. Through a combination of biochemical and biophysical assays, we identified two potent non-nucleoside compounds with activity against our PicoGreen beta-coronavirus panel. Mechanism of action investigations revealed these compounds bind exclusively to the nsp12-8 complex, unveiling a potentially unique inhibitory mechanism. These compounds serve as valuable starting points for structure-activity relationship (SAR) explorations and potential therapeutic leads.