Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified -hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elder adults 1 . Neither a vaccine nor an effective antiviral therapy exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the nucleoprotein-RNA (N-RNA) template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase (PRNTase) and cap-specific methyltransferases (MTases) activities 2,3 . How P interacts with L and mediates association with the free form of N and with the ribonucleoprotein (RNP) is not clear for HMPV or other prominent human pathogens including Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of>1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.