The continuous evolution of influenza A strains and limited efficacy of the current anti-influenza drugs highlights the need for new anti-influenza therapeutics. Influenza uses a process called `cap snatching' where the viral polymerase binds to a host pre-mRNA cap (m7GpppNp), and then the viral endonuclease cleaves the mRNA 11-13 nucleotides downstream of the cap. The goal of this grant is to develop inhibitors for the highly- conserved endonuclease, an essential molecular target for viral replication. 5-bromo-3-hydroxypyridin(1H)-2-one was identified as a promising inhibitor of influenza A endonuclease from a X-ray crystallographic screening campaign of 775 small molecule fragments. This scaffold was optimized to greater than a 1000-fold potency using an integrated platform combining (i) medicinal chemistry, (ii) fluorescence-based high-throughput endonuclease assay, (iii) X-ray crystallographic analyses, (iv) in silico modeling, and (v) in vitro antiviral assays, including a new semi-high throughput antiviral fluorescence-based assay that is efficient for rapid evaluation of new compounds for anti-influenza activity. Current lead compounds have endonuclease inhibition of less than 16 nM compared to 16 M for the initial hit. Several compounds demonstrated viral inhibition in MDCK cells with an EC50 value of 2.5 M with cytotoxicity (CC50) greater than 50 M. In this R21/R33 application, we propose synthesis of second generation 3-hydroxypyridinones by further structure-based optimization to (i) continue to strengthen viral inhibition to sub-micromolar concentrations, (ii) ensure minimal cytotoxicity, (iii) examine and improve efficacy of the proposed compounds by testing and optimizing against potential resistant mutant variants, and (iv) optimize the compounds for in vivo efficacy in a mouse model system. Based on the established structure-based optimization path and opportunities to grow the current 3- hydroxypyridin(1H)-2-one lead molecule to interact with other parts of the binding pocket, we are confident of developing suitable drug-like molecules within the two-year R21 project period. Two or three optimized compounds (R21) will be further optimized for pre-clinical absorption, distribution, metabolism, excretion, and pharmacokinetics (ADME/PK) evaluation and animal model studies in the R33 phase.