Discovery and Optimization of a Novel Series of Influenza A Virus Replication Inhibitors Targeting the Nucleoprotein Protein–Protein Interaction

Influenza A virus (IAV) is a negative-sense, single-stranded RNA virus that causes seasonal epidemic respiratory infections, with novel subtypes of IAV historically able to lead to pandemics that spread on a global scale. We conducted a phenotypic high-throughput screen (HTS) that identified compound 1 as a singleton hit. Resistant viral mutants generated against analog 2 revealed mutations in the nucleoprotein (NP). An X-ray cocrystal structure of NP in complex with compound 3 helped define the novel mechanism of action as disruption of the NP-NP protein-protein interaction (PPI), leading to inhibition of NP oligomerization and blocking viral replication. Medicinal chemistry optimization efforts resulted in the identification of compound 20 (VNT-101) as a potent IAV inhibitor with low nM activity across multiple subtypes. Compound 20 has attractive DMPK and physicochemical properties, and demonstrated robust antiviral activity in rodent models of influenza infection, leading to successful completion of IND-enabling safety studies.