In the continuing absence of an effective vaccine, topical microbicides offer a credible alternative preventive strategy to reduce sexual transmission of HIV-1. Several viral fusion and entry inhibitors have been shown to prevent SHIV infection of rhesus macaques by the vaginal and/or rectal routes and are in preclinical and early clinical development as microbicide candidates. HIV-1 membrane fusion is mediated by a series of large-scale structural transitions in the gp41 envelope glycoprotein. Evidence indicates that a transient gp41 species known as the prehairpin intermediate is a potential target for drugs that inhibit HIV-1 entry. The long- term goal of this research plan is to use modern molecular and structural methods to identify and develop a novel small-molecule gp41 fusion inhibitor for inclusion in a topical HIV-1 microbicide. To achieve this, we will capitalize on specific surface features revealed by our recent structure determination of an autonomously folded, trimeric coiled-coil subdomain of gp41 that provides an atomic model for the putative prehairpin conformation, as well as small-molecule lead compounds developed by means of an innovative structure-based drug design technology. We propose the following specific aim for the R21 component of this project: 1) To identify and optimize two series of novel small-molecule compounds that inhibit HIV-1 membrane fusion by targeting the gp41 prehairpin intermediate. We will design and synthesize two sets of analogs of active triazinone and biphenyl compounds, characterize the equilibrium properties of interactions with the N-trimer coiled coil, and evaluate their anti-HIV-1 activity and mechanism of action. Bound inhibitors will be visualized by x-ray crystallography in order to allow refinement of binding affinity. The specific aims of the R33 phase of the project are: 2) To characterize the specificity, potency and toxicity of improved small-molecule compounds with enhanced gp41 inhibitory activity. We will conduct in vitro studies in primary cells and human cervicovaginal tissue explants to determine the virucidal activity of select small- molecule gp41 inhibitory compounds against diverse primary HIV-1 isolates, and their potentially toxic or inflammatory effects. We will also use the rabbit vaginal irritation model to evaluate the irritation potential of the fusion inhibitors. 3) To assess the in vivo potency and breadth of activity of optimized small- molecule fusion inhibitors alone and in combination with entry inhibitors targeting HIV-1 gp120 (BMS- 378806) and CCR5 (CMPD167) using the NOD/SCID-hu BLT mouse vaginal transmission model. We will evaluate the protection of humanized BLT mice from vaginal challenge with multiple HIV-1 variants by small- molecule fusion inhibitors alone and in synergistic combination with BMS-378806 and CMPD167. Our emphasis is to identify a new class of potent HIV-1 fusion inhibitors suitable for development as a component of a microbicide formulation.