This proposal focuses on the development of entry inhibitors against Human Immunodeficiency Virus Type-1, by targeting the viral fusion protein gp41. Gp41 mediates fusion to mammalian host cells via a highly conserved coiled coil domain. The hypothesis is that compounds that bind to this domain will act as fusion inhibitors by preventing the gp41 conformational rearrangement required for fusion. Discovery of inhibitors to the gp41 coiled coil is hampered by the fact that this domain is not readily accessible in solution, being buried in intact gp41 and unstable when excised from the rest of the protein. We have developed a stabilized coiled coil construct through the use of bidentate bipyridyl groups covalently attached at the N-termini of gp41 peptides. The addition of transition metal ion to the bipyridylated peptides stabilizes the trimeric helical structure of the coiled coil, through the formation of an octahedral tris-bipyridyl complex, and removes non-specific aggregation of the hydrophobic peptide. The goal of this proposal is to make use of the probe properties of various metal ions to establish a screening and structural assay for inhibitors which bind to the coiled coil. 1) Discrete metal ion - coordinated constructs which represent the gp41 inner coiled coil will be developed and analyzed by NMR structure determination. 2) Constructs will be used in the development and refinement of a fluorescence binding assay to detect compound binding to the coiled coil. 3) Library screening will be conducted and successful hits analyzed with in vitro syncytium and viral infectivity assays. The metal ion used in each of these specific aims will be selected to have diamagnetic, paramagnetic or fluorescent properties, as needed, to be utilized in the biophysical assays. The long term goal of this proposal is to develop promising non-peptide drug candidates for HIV-1 fusion inhibition.
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