Design and characterization of HIV fusion inhibitors targetinggp41

The complex process of entry of the human immunodeficiency virus (HIV) into a target cell is an attractive target for anti-HIV drug design. Three classes of currently existing fusion inhibitors target the ectodomain of gp41 in the prehairpin intermediate state, thereby preventing formation of the fusogenic trimer of hairpins. In an attempt to broaden the spectrum of the activity of these inhibitors against diverse HIV strains as well as to optimize their inhibitory properties, variability of ectodomain of gp41 from distant viral strains was analyzed and used to select representative strains from distant viral lineages. Envelope sequences for further studies were selected from three strains of HIV-1, two strains of HIV-2, and one strain of SIV.; The spectrum of inhibition of human (HIV) and simian (SIV) immunodeficiency virus envelope (Env)-mediated cell fusion by C34, a 34-residue peptide corresponding to the C-heptad repeat of gp41 (residues 628--661 of HIV-1 Env), has been examined using a panel of five envelope glycoproteins, three from HIV-1 viruses and two from SIV, and six C34 peptides derived from selected representative strains. The inhibition data from the panel of 30 C34/envelope glycoprotein combinations span IC50 values ranging over four orders of magnitude and permit one to rationalize both the potency and broadness of the inhibitory properties of the C34 peptides.; Chimeric construct N35CCG-N13) comprising exposed trimeric N-terminal helices of gp41 was changed based on the sequences of N-terminal heptad repeats of the representative strains and the resulting N36 construct was cloned, expressed and purified. The fusion inhibitory activity of the resulting chimeric proteins was analyzed using a quantitative vaccinia-virus based reporter gene assay for inhibiting cell fusion, mediated by HIV envelope derived from different HIV and SIV strains, as well as live virus infectivity assay in human peripheral blood mononuclear cells.; Positions e and g of the helical wheel from the N-helices are critical for interaction with the C-helices in the fusogenic form of gp41. It was previously shown that a peptide, N36 Mut[e,g], in which all the residues at positions e and g of the helical wheel were mutated to prevent any interaction with the C-helix, inhibits fusion ∼50 fold more effectively than the native N36 peptide from which it was derived. Combinations of N35CCG-N13 chimeric protein and N36Mut[e,g] peptide were analyzed using cell-cell fusion assay and found to be synergistic. This finding provides new insights into the mechanism of inhibition of membrane fusion, as well as a new potential direction for fusion inhibitor design.