While anti-retroviral therapy (ART) can successfully control virus replication in HIV-1-positive individuals, the virus is suppressed rather than truly eradicated. Furthermore, toxicity and drug resistance associated with long- term ART remain a significant challenge for effective AIDS therapy. Thus, there is a need to develop new therapeutics with novel mechanisms of action to improve current ART. As a step toward developing novel anti- HIV agents, we have identified a class of quinolizidines, including aloperine, which inhibit HIV at 1-5 uM by blocking viral entry. Aloperine has been shown to exhibit anti-inflammatory properties, which could potentially be beneficial for relieving immunopathogenesis associated with HIV-1 infection. Our preliminary structural optimization has yielded aloperine derivatives with approximately 15-fold increase in anti-HIV-1 activity. Our mechanism of action study reveals that the V1/V2 loop of gp120, which is the target of several broad and potent neutralizing antibodies, is a critical determinant of the anti-entry activity of a current lead aloperine derivative. Based on these promising results, we hypothesize that potent anti-HIV quinolizidines can be obtained through rational drug design and become anti-HIV-1 drug candidates with novel mechanisms of action. The goal of this research project is to improve the anti-HIV potency of aloperine derivatives and determine their pharmacological profiles, such as mechanism of action and the breadth of anti-HIV activity, which are essential for further drug development. The following Specific Aims will be carried out to test this hypothesis and achieve our goal in identifying promising anti-HIV entry inhibitors: 1) to elucidate the molecular mechanisms of action of the anti- HIV-1 entry activity of quinolizidines; 2) to identify potent quinolizidine derivatives through lead optimization; 3) to establish the anti-HIV-1 profiles of aloperine derivatives. Aloperine (MW=232) is a natural product found in several common plant species and can be obtained by total synthesis. Aloperine has been shown to have optimal PK profiles and to be effective in animal models of inflammatory diseases. Thus, it is an attractive hit for lead optimization. Such compounds with the dual modes of action may become a useful addition to ART, and have potential to reduce inflammation associated with persistent HIV-1 infection. We plan to complete the study in three years to reach the following milestones: a) obtaining aloperine derivatives with low nM potency, b) elucidating a novel mechanism of how these compounds inhibit HIV-1 entry, and c) identifying aloperine derivatives that exhibiting cross clade anti-HIV-1 activity and optimal pharmacokinetic profiles. Completion of the work in this proposal will yield highly promising anti-HIV1 agents and is critical for their further preclinical development including animal efficacy studies and optimization of their anti-inflammatory activity.