Principal Investigator/Program Director (Last, First, Middle): Chaikei"), Irwin M DESCRIPTION: The overarching goals of this research project are to identify mechanisms of HIV-1 cell entry antagonism and derive structure-based antagonists. HIV-1 infection is dependent on attachment and entry of the virus into host cells. Virus-cell recognition occurs through initial interactions of viral spike envelope protein, gp120, with CD4 and a co-receptor, most commonly a chemokine receptor CCR5 or CXCR4. Interactions of gp120 with these receptors induce rearrangements in gp120-gp41, leading to exposure of structural components of gp41 required for virus-host cell membrane fusion. We now know that the multiple interactions of HIV-1 gp120 influence each other cooperatively, leading to progressive structural changes from flexible unliganded states to more conformationally organized forms with matured binding sites. We believe that molecules that can bind to gp120 and disrupt receptor-induced gp120 structuring can effectively disrupt the multiple receptor and gp41 interactions of gp120, and in this way, lead to effective pathways for entry antagonism. In this part of the Program Project, we will advance a miniprotein/peptide based strategy to identify molecular compounds that can suppress gp120 receptor site binding and structuring and lead to antagonists of HIV-1 entry. There are four specific aims. Aim 1. Using miniprotein and peptide starting points, identify gp120 antagonists that inhibit binding of HIV-1 Env protein to CD4 and co-receptor sites through targeting both the conserved CD4 binding site and allosteric sites. Aim 2. Map binding sites of peptide antagonists in gp120 and use this information as a guide to design higher- potency, specific antagonists as well as to more fully elucidate neutralizing sites in gp120 as long-term targets for entry inhibition. Aim 3. Profile inhibition patterns of gp120 monomers, trimers and Env mutants with peptide antagonists, and identify structural features of env protein that limit the efficacy of peptide-derived inhibitors. Identify modified peptide inhibitors with improved inhibition profiles against trimeric forms of HIV-1 envelope. Aim 4. Convert allosteric and competitive miniprotein and peptide antagonist leads into structure-minimized antagonists of HIV-1 Env with optimized dual antagonist effects in molecular assays and optimized potencies in cell infection assays. Evaluate their potential to lead to small-molecule antagonists of entry and as functional tools to elucidate mechanisms of gp120 interactions. This project will help to identify molecular approaches to control and ultimately antagonize the cooperative interactions that drive HIV-1 cell entry. The results will help amplify protein interaction mechanisms of the virus- cell entry machine. More generally, this work will help identify methods for designing therapeutic agents for flexible protein targets known to occur in other viruses.