Project Summary Despite the existence of vaccines and antiviral drugs, global annual death tolls attributed to influenza virusinfection are ~500,000. Currently there is only one orally bioavailable drug, oseltamivir, that is still in use totreat influenza infection. The alarming fact is that oseltamivir-resistant influenza strains have already beenisolated from human patients, and several of them appear to have adapted the fitness of transmission.Thus there is a pressing need to develop novel antivirals to combat these drug-resistant influenza viruses.They can be used either alone to inhibit oseltamivir-resistant strains or in combination with oseltamivir todecrease the pace of drug resistance evolution. Moreover, as the influenza viruses circulating amonghumans consist of at least two influenza A strains (H1N1 and H3N2) and two B strains (Yamagata andVictoria), it is also desirable to have one antiviral drug with broad-spectrum antiviral activity against all fourstrains. In response to the need for a next generation of antiviral drugs with broad-spectrum antiviralactivity, especially against multidrug-resistant influenza viruses, we performed an in silico screening of anin-house library of small molecules predominantly prepared by one-pot multicomponent reaction(MCR) methodologies against the virus polymerase PA subunit and have identified several promising hits. Onecompound, UAWJ85, inhibits several multidrug-resistant influenza A and B viruses with EC50 values rangefrom single to sub-micromolar. This compound also displays a high in vitro genetic barrier to drugresistance, as no resistant viruses were selected after 10 passages with increasing concentrations of thecompound. Mechanistic studies confirmed the inhibition of polymerase PA-PB1 subunit interactions byUAWJ85. The broad-spectrum antiviral activity and high in vitro genetic barrier to drug resistance ofUAWJ85, coupled with the expeditious structure?activity relationship studies using the one-pot Ugi-Azide 4CR methodology, have led us to further optimize the antiviral potency, selectivity index, in vitro and invivo PK properties of lead compounds and test their in vivo antiviral efficacy in mice. In Aim 1, we will optimize the in vitro antiviral and pharmacokinetic properties of UAWJ85. A listof criteria was imposed for compound progression. The goal is to prioritize lead compounds for in vivomice studies. In Aim 2, we will will test the in vivo PK and antiviral activity of UAWJ85 or its analogs usingthe influenza virus infected mice model. In summary, the advantage of exploring MCR products for broad-spectrum anti-influenza drugs has beenclearly demonstrated by the preliminary results. This proposal, if successfully implemented, will lead to theurgently needed antivirals to combat both seasonal outbreaks and the next influenza pandemic.