Validation of Small-Molecule Entry Inhibitors Targeting the Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections worldwide, particularly in infants, young children, and elderly individuals. Despite the availability of preventive options such as vaccines and monoclonal antibodies, effective antiviral therapies for acute infections remain limited. Therefore, we investigated small-molecule entry inhibitors identified in a high-throughput campaing and evaluated the most promising candidates in detail by performing resynthesis and determining their antiviral activity. Unfortunately, analytical investigations by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) revealed discrepancies between the screening samples and the resynthesized compounds for two compound classes, highlighting the complexity of hit validation and the potential influence of impurities. By publishing these results, we aim not only to underscore the importance of rigorous analytical and biological validation in screening campaigns but also to support the development of improved strategies for identifying reliable RSV inhibitors. We also believe it is important to publish false-positive hits to avoid time efforts on projects that assume such compounds as genuine hits.