A Parallel Phenotypic Versus Target-Based Screening Strategy for RNA-Dependent RNA Polymerase Inhibitors of the Influenza A Virus

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.