Developing Inhibitors of Seasonal and Pandemic Influenza Viruses

Summary Influenza viruses are a leading cause of human disease due to respiratory viral infection worldwide. It is the overarching objective of this project to advance prodrug conjugates of 4'-fluorouridine (4'-FlU), a novel pyrimidine analog with confirmed broad oral efficacy against influenza viruses, and at least one non-nucleoside inhibitor of the influenza virus polymerase complex, to the stage of formal preclinical development and assembly of an investigational new drug-enabling package. The project design is driven by our underlying hypothesis that effective next-generation therapeutics for the treatment of influenza must be orally available, display a broad indication spectrum against influenza virus isolates of human, avian, and swine lineages, and ideally cover both influenza A (IAV) and B (IBV) viruses. These product profile demands are derived from the clinical burden imposed by the diverse spectrum of seasonal influenza viruses, the pandemic potential arising from spillover of zoonotic avian viruses into the human population, and current FDA recommendations that recognize outpatient adults suffering from seasonal influenza as the primary patient population for initial clinical testing. These developmental objectives are best met with direct acting therapeutics, since host-targeted antiviral therapies, although often tantalizingly broad in indication range, are prone to unacceptable side effects that are incompatible with the primary patient group pursued. In previous work underpinning this program, we have demonstrated broad-spectrum antiviral activity of 4'-FlU in cultured cells and animal infection models against a clinically significant panel of RNA viruses. Against the influenza virus indication, 4'-FlU has confirmed oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in rodent and non-rodent models. The compound triggers immediate chain termination of the influenza virus RNA-dependent RNA polymerase (RdRP) complex and carries a high barrier to viral resistance. In preparation of clinical development, we have generated prodrug conjugates of 4'-FlU to optimize delivery across the gastrointestinal epithelium. To broaden our influenza virus RdRP inhibitor portfolio, we have identified a set of non-nucleoside RdRP inhibitors in a large-scale high-throughput screening campaign that will be advanced simultaneously as companion drugs or for combination therapy in a multi- pronged approach. To select a clinical candidate for formal development, 4'-FlU prodrug conjugates will be subjected to de-risking efficacy and tolerability testing in relevant rodent and non-rodent animal models and dosing paradigms explored through interfacing of dynamic PK profiles with performance in human airway epithelium organoids (aim 1). Non-nucleoside RdRP inhibitor lead candidate scaffolds will be synthetically developed, mechanistically characterized, and subjected to proof-of-concept efficacy testing (aim 2). Emerging non-nucleoside leads will be queried for combination therapy with the 4'-FlU clinical candidate and the effect of mono- and combination therapy on suppressing influenza virus transmission determined in ferrets (aim 3).