Identification and characterization of a novel inhibitor of influenza A virus that acts by blocking nucleoprotein oligomerization

Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of acute respiratory infections resulting in significant morbidity and mortality. Although approved influenza antivirals (e.g., oseltamivir and baloxavir) exist, concerns persist about the potential for emergence of drug-resistant variants, highlighting the continuing need for new antiviral therapies. Here, we describe the development of an orally bioavailable, direct-acting antiviral (VNT-101) with a novel mechanism of action: disrupting homo-oligomerization of the influenza nucleoprotein (NP) and thereby inhibiting viral RNA synthesis. Selection of drug-resistant mutants revealed amino acid substitutions mapping to the oligomerization domain of NP, and X-ray crystallography co-structure determination of VNT-101 complexed with recombinant NP confirmed VNT-101 binding in the oligomerization pocket. Biochemical experiments using size exclusion chromatography confirmed disruption of oligomerization when this chemotype is added to preparations of recombinant NP <i>in vitro</i>. VNT-101 has potent and specific activity against the currently circulating IAV subtypes H1N1 and H3N2, with mean EC₅₀ values ranging from 2 to 18 nM, and displays strong efficacy in a murine model of lethal influenza infection when administered either prophylactically or therapeutically. Importantly, VNT-101 remains active against influenza variants that are resistant to either oseltamivir or baloxavir and also has potent activity against highly pathogenic avian H5N1 and H7N9 isolates that have transmitted to humans and represent strains of potential pandemic concern. These studies support the continued development of VNT-101 to augment our therapeutic arsenal against both seasonal and pandemic influenza.