Targeting G3BP1-Mediated Stress Granules to Suppress SARS-CoV-2 Replication.

Stress granules (SGs) are cytoplasmic, membraneless assemblies of proteins and RNAs that transiently form in response to cellular stress. Dysregulation of SGs has been associated with cancer, neurodegeneration, and viral infection. GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a central SG component that regulates granule assembly and dynamics through protein-RNA and protein-protein interactions (PPIs). During viral infection, SGs can be hijacked to enhance viral replication and suppress host defense. In this study, we show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N) binds G3BP1 and localizes to G3BP1-positive SGs in infected cells. We hypothesize that small-molecule ligands targeting G3BP1 can disrupt the G3BP1-N interaction and reduce viral replication. To test this, we developed a robust, miniaturized, 384-well time-resolved fluorescence resonance energy transfer (TR-FRET) assay for high-throughput screening (HTS). Screening a library of 2560 FDA-approved drugs yielded 17 hits. Subsequent counterscreening, validation, and biophysical binding studies identified 7 PPI inhibitors with IC