Disruption of lipid bilayers is an obligatory step in virus:cell fusion, the process that delivers the viral genome into the host cell. Some of the domains of the HIV- 1 transmembrane glycoprotein (TM) that mediate virus:cell fusion have been targeted for antiviral drug development, however, combinatorial design of drugs against membrane-interactive domains of TM could be facilitated with an effective screening assay. We recently developed a microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules. The assay is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb3+) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb3+ is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with UV light. For the microwell plate assay, large unilamellar phospholipid vesicles (LUV) that have either Tb3+ or DPA entrapped and the opposite molecule in the external solution are prepared. Disruption of the membranes allows the Tb3+/DPA complex to form, giving rise to a fluorescent solution. Preliminary results indicate that a synthetic peptide corresponding to amino acids 828-855 at the carboxyl terminus of TM, known as the lentivirus lytic peptide one (LLP-1), has vesicle lysing activity in the picoMolar (pM) concentration range. This synthetic peptide has at least 100 times greater activity in this assay than melittin, a naturally occurring cytolytic peptide from bee venom. Studies proposed using LLP synthetic peptides will test our hypothesis that this assay is useful in screening for membrane disruptive domains of TM (Specific Aim 1). We will also establish if synthetic peptides corresponding to other domains of TM cause membrane perturbations detectable by this assay (Specific Aim 2) and determine if the assay can be used to develop potentially novel classes of human monoclonal antibodies which bind motifs in the TM ectodomain and block their interactions with membranes (Specific Aim 3). The proposed studies will provide proof-of-concept that the Tb3+/DPA:LUV assay is useful for rapidly identifying and characterizing inhibitors of membrane disruptive domains of HIV-1. The approaches piloted here may be useful for developing drugs against the AIDS virus and a broad range of important human viral pathogens with similar fusion proteins. [unreadable] [unreadable]