DESCRIPTION: The goal of this proposal is the optimization of newly discovered HIV-1 entry inhibitors and elucidation of their broad mechanism of action. HIV-1 infection begins when the envelope glycoprotein gp120 binds to the cell surface receptor CD4. This initial binding event results in a series of allosteric events from activation of the co- receptor site, subsequent binding to the chemokine co-receptor (CCR5 or CXCR4), fusogenic conformation of gp41, and finally fusion of the viral and cell membranes. As the process of entry is key to the replication o the virus, it is one of the most important targets in the search for new drugs to treat HIV-1 infection. Advances in knowledge of the molecular mechanisms of HIV-1 entry have allowed the discovery and development of molecules that block each step of the entry process and that have been successfully used in the clinic, e.g., maraviroc (Selzentry, Pfizer, New York, NY) and enfuvirtide (Fuzeon, Hoffman-La Roche, Nutley, NJ). To date, however, no gp120-targeted therapies have been approved for use in the clinic. Using an innovative high- content pharmacophore approach, we have discovered SC03, a new entry inhibitor that shows micromolar potency in a single-round infection assay. Bioisosteric replacement of a central piperazine and an acenaphthene moiety in this compound has led to the identification of compound SC08 - a novel entry inhibitor with a new chemotype and sub-micromolar potency. We will combine medicinal chemistry with computer-aided drug design (CADD) and biological testing for potency, selectivity, and toxicity. Our approach integrates high- content pharmacophore mapping and three-dimensional quantitative structure-activity relationships, chemical synthesis, direct binding assays using recombinant wild-type and mutant HIV gp120 variants, and single-round infection assays using virus pseudotyped with envelopes from genetically disparate primary HIV isolates. The toxicity of compounds will be tested using uninfected PBMCs as well as 293T and U87 cells to assess therapeutic index. The success of this project will provide novel potent and selective HIV-1 entry inhibitors that may ultimately expand the anti-HIV armamentarium.