Respiratory syncytial virus two-step infection screen reveals inhibitors of early and late life cycle stages

Screening 6,048 molecules, we identified 23 primary candidates of which 13 were active in the first round and ten scored preferentially in the second round of infection. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited hRSV transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses including hCoV-229E. One compound, identified in the 2nd round of infection, neither measurably inhibited hRSV cell entry nor hRNA replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated l BCi-NS1.1 lung cells.In total, we identified four new hRSV inhibitor candidates with different modes of action. A two-step screening system, as used here, may be useful for the discovery of new chemical entities inhibiting hRSV via diverse mechanisms and targets.