Identification of small-molecule inhibitors of the SARS-CoV-2 RBD–ACE2 interaction by structure- and affinity-based screening

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein mediates viral entry through its interaction with angiotensin-converting enzyme 2 (ACE2), making it an attractive target for antiviral intervention. However, the broad and relatively shallow ACE2-binding interface presents a challenge for small-molecule inhibitor discovery. In this study, we explored conserved regions outside the ACE2-binding interface as potential druggable sites for small-molecule inhibitors targeting the SARS-CoV-2 RBD. Structure-based virtual screening was performed using the structure of the SARS-CoV-1 RBD as a structural surrogate, and selected compounds were evaluated using a newly established affinity-based assay on diazirine-modified amorphous carbon plates. Evaluation of 175 virtual hits identified eight RBD-binding compounds. Among them, only RABI-1 inhibited RBD-ACE2 interaction in a Calu-3 cell-based binding assay without affecting ACE2 expression or cell viability. Evaluation of structural analogs of RABI-1 led to the identification of RABI-3, which showed stronger inhibitory activity compared with RABI-1. In addition, RABI-3 inhibited the interaction between ACE2 and RBDs derived from multiple SARS-CoV-2 variants in an AlphaLISA immunoassay and suppressed pseudovirus infection in Calu-3 cells. These findings support the feasibility of targeting regions of the RBD outside the ACE2-binding interface as a strategy for identifying small-molecule inhibitors of SARS-CoV-2 entry.