Influenza is a contagious respiratory illness caused by influenza virus, which can cause mild to severe illness, and at times can lead to death. Currently, the neuraminidase (NA) inhibitors, oseltamivir, zanamivir, and peramivir, are the FDA-approved anti-Influenza virus drugs recommended by the US CDC for use against recently circulating influenza viruses. However, there are many different flu virus subtypes and they are constantly changing. This is further compounded by emerging drug resistance and limited effectiveness associated with anti-flu drugs. Thus, novel and effective antiviral agents are needed to cope with influenza. The long-term goal of this study is to identify novel anti-influenza virus agents with potent and broad activity to control flu virus infection. As a step toward this goal, we have identified a class of quinolizidines, such as aloperine, that inhibit multiple subtypes of influenza viruses including a CDC panel of NA inhibitor resistant influenza A and B viruses at sub-uM concentrations. Targeting the viral nucleoprotein (NP) appears to be responsible for the broad anti-influenza virus activity of the quinolizidines. More importantly, a lead aloperine derivative is effective in vivo using a mouse model. With these promising results, the goal of this research project is to improve the potency of the quinolizidines and to elucidate their mechanism of action. The working hypothesis is that through rational drug design, quinolizidine derivatives with potent and broad anti-influenza virus activity can be obtained. Two Specific Aims will be carried out to test this hypothesis and achieve our goal in identifying promising anti-influenza virus agents. Aim 1 is to improve the potency of quinolizidines by structural modifications. Our approach to achieve this aim is to optimize the functional groups of the quinolizidines to obtain derivatives with low nM potency and optimal pharmacological profiles. Aim 2 is to establish the antiviral profiles and to elucidate the mechanism of action of the quinolizidines. The goal of this aim includes determining the pharmacological profiles and defining mechanisms of action of the quinolizidines. Pharmacological evaluation will focus on determining the breadth of antiviral activity and the efficacy of quinolizidine derivatives in a mouse model. The hit compound, aloperine, is a quinolizidine with unique physicochemical and biological properties suitable for lead optimization. It has been tested in cell and animal models for its therapeutic effects in ulcerative colitis and regulation of inflammatory cytokines. Our preliminary lead optimization efforts have resulted in derivatives with sub-uM activity. Completion of the proposed study is expected to yield new anti-flu agents that inhibit a broad spectrum of influenza viruses, including viral strains that are resistant to the current CDC recommended anti-influenza therapeutics, at low nM potency.