Inhibition of HIV Infection by Pseudopeptides Blocking Viral Envelope Glycoprotein-Mediated Membrane Fusion and Cell Death

Abstract

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.

Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

“…The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5[KPR]-TASP molecules, manifested maximum inhibitory activity on infection of cells by HIV-1 and -2 but not by simian immunodeficiency virus isolates, thus emphasizing the specific nature of these TASP inhibitors. In already infected cells, the TASP inhibitors also blocked syncytium formation and the occurrence of apoptosis (7). These observations indicated that these TASP inhibitors block a defined target that should be implicated in the functioning of the gp120⅐gp41 complex to initiate viral entry, syncytium formation, and apoptosis.…”

“…Structure and inhibitory activity relationship studies using analogs of 5[KPR]-TASP indicated that the two basic residues Lys and Arg in the tripeptide are essential and can be replaced by each other and that their positively charged side chains play a critical role in the inhibitory structure. Interestingly, replacement of Leu amino acid residues by Asp amino acids or the reduction of the peptide bond between the first two amino acids of the tripeptide generated pseudopeptide-TASP analogs active at submicromolar concentrations on HIV infection (7). By the use of FITC-and biotin-labeled 5[K⌿(CH 2 N)PR]-TASP constructs, we demonstrate here that the TASP inhibitors bind specifically to a 95-kDa, cell surface protein (p95).…”

“…In view of this, we have designed and synthesized a construction referred to as template assembled synthetic peptide (TASP) in which templates such as KKKGPKEKGC and KKKKGC were employed to covalently anchor arrays of tripeptides (RPR, RPK, or KPR) at the ⑀-amino group of the lysine residues in the templates (7). The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5[KPR]-TASP molecules, manifested maximum inhibitory activity on infection of cells by HIV-1 and -2 but not by simian immunodeficiency virus isolates, thus emphasizing the specific nature of these TASP inhibitors.…”

“…The identity of p95 remains to be elucidated. However, because the TASP inhibitors block both HIV envelope-mediated virus to cell and cell to cell membrane fusion processes and the initiation of apoptosis (7), it can be suggested that p95 is implicated as a potential cofactor of CD4 in the functioning of the gp120⅐gp41 complex to initiate membrane fusion and thus viral entry. Consistent with this, 5[K⌿(CH 2 N)PR]-TASP blocks the binding of HIV particles to CD4 ϩ permissive cells at the same efficiency as an anti-CD4 monoclonal antibody.…”

Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin

“…The expression of the nucleolin at the cell surface was illustrated by using a specific antagonist of nucleolin, the HB-19 pseudopeptide that was designed initially as a potent inhibitor of HIV infection [13][14][15][16]. HB-19 presents pentavalently the tripeptide KPR in which the peptide bond between the K and P is reduced in order to increase the endopeptidase resistance of this pseudopeptide.…”