Screening And Identification Of Short Cyclic Peptides Interacting With The HIV-1 Gp41 NHR And Mimicking The Epitopes On The Fusogenic Gp41 Core Structure.

Acquired immunodeficiency syndrome (AIDS), one of the serious emerging infectious diseases worldwide, is caused by the human immunodeficiency virus (HIV).The HIV envelope glycoprotein (Env) plays an important role in HIV entry into the target cell. The Env surface subunit is responsible for virus binding and the transmembrane subunit gp41 mediates fusion between the viral and target cell membranes. HIV-1 infection is initiated by gp120 binding to CD4 and a co-receptor (i.e., CXCR4 or CCR5) on target cell, which triggers a conformational change in gp41. The interaction between the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of the gp41 extracellular domain leads to the formation of a six-helix bundle (6-HB), representing the fusion-active gp41 core. Peptides derived from the CHR region, named C-peptides, e.g., C34 and T-20, inhibit HIV fusion by interacting with the viral NHR region and blocking the fusogenic core formation. Therefore, gp41 NHR region can serve as an attractive target for developing HIV fusion inhibitors, a new class of anti-HIV drugs and the 6-HB formed by peptides derived from the NHR and CHR regions can be used as a model of the gp41 core to study fusogenic mechanism of HIV and the mechanism of action of the anti-HIV peptides.Accordingly, the main aims of this proposal are: (1) To screen short peptides with inhibitory activity on HIV-1 entry from phage display libraries as leads for developing peptidic HIV fusion inhibitors;and (2)To identify the epitopes on gp41 core structure and the epitope which can bind gp41 NHR.1.Screening the C7C phage library using soluble form of biotinylated peptide N36 derived from gp41 NHR region (PL+S method). In this assay, the positive clones binding to biotin-N36 in solution were captured by streptavidin-coated on the solid phase. After three cycles of biopanning, we identified 16 clones which may be the mimotopes of peptide C34 since the binding of the positive clone No.8 to the peptide N36 could be blocked by peptide C34 in a competitive ELISA.Amino acid sequences of the displayed peptides in 10 phage clones were deduced from DNA sequences.They are CQHWWHWYC, CVHWWHWIC, CYWWHRLHC, CTWWRPWHC, CHPHWWRTC, CWYWHAKHC, CFWWHTRHC and CPAPDQSMC. Every peptide sequence contains at least two hydrophobic residues. 9 of the 10 clones have a conservative sequence WW, which may mimic the critical conserved hydrophobic residues W628 and W631 in the CHR cavity-binding sequence to interact with the NHR cavity-forming sequence.2. Screening of the C7C phage display peptide library using the peptide N36 coated on the solid phase (P+LS method). In this assay, the positive clones were captured by the biotin-N36 bound to the streptavidin-coated wells. After three cycles of biopanning, we identified 11 positive phage clones. The binding of the positive clones to N36 could be inhibited by C34 and ADS-J1, a small molecule HIV fusion inhibitor targeting gp41 NHR region. All the positive clones showed a unique amino acid sequence: CDRHQHKRC.3. Screening C7C phage display peptide library using polyclonal antibodies against the gp41 heptad repeat regions. Thirteen positive phage clones were identified and 8 of 13 clones could bind to N36 peptide. All these 8 positive clones shared a common sequence: CPYGPKLC, suggesting that CPYGPKLC may be a gp41 NHR binding sequence.1. The peptide FJ-08-Ex corresponding to the sequence of the positive clone No.8 (SACYWWHRLHCGGGS) identified from the PL+S screening method was synthesized. The natural sequences SAC and CGGGS were added to the N- and C-termini, respectively, of the core sequence YWWHRLH in order to keep a stable cyclic conformation, which may be required for its interaction with the gp41 NHR region. Compared with unrelated control peptide 13b, FJ-08-Ex significantly inhibited HIV-1 mediated cell-cell fusion during the first 2 hrs of coculture of HIV-1 infected cells and the target cells in a dose-dependent manner. However, the inhibitory activity disappeared 24 hrs later.