Characterization of novel respiratory syncytial virus inhibitors identified by high throughput screen

•Three novel RSV inhibitor series were identified by RSV replicon HTS.•Each series involved a distinct mode of action and resistance.•Provided tool compounds to advance RSV antiviral discovery and viral replication research. Respiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infections with no effective treatment available. Finding novel inhibitors of RSV is an important first step towards developing an efficacious RSV therapy. Here we report the characterization of three novel classes of RSV replication inhibitors identified through a high throughput RSV replicon screen of ∼1million compounds in the AstraZeneca compound collection. These inhibitors, cpd 1, 2, and 3, specifically targeted RSV and were not active against other viruses tested. Resistance selection in RSV A2 with cpd 1 identified escape viruses with mutations mapped to the RSV L protein, an RNA-dependent RNA polymerase (Y1631C and I1413T). Recombinant RSV containing the L Y1631C substitution conferred resistance towards cpd 1, suggesting that the RSV polymerase is the target of this inhibitor. Interestingly, cpd 3, a nucleoside analog, induced a single resistant mutation in the P protein (D231V), indicating a novel mode of action not previously reported. cpd 2 affected host cell cycle and no frequent mutation was isolated following resistance selection, suggesting its possible involvement of a host-targeted mechanism. Taken together, we have identified three novel RSV inhibitors with different modes of action, providing new chemistry starting points for the discovery and development of future RSV therapeutic treatment.