Design of potent inhibitors of HIV-1 entry from the gp41 N-peptide region.

The HIV-1 gp41 envelope glycoprotein promotes fusion of the virus and cell membranes through the formation of a trimer-of-hairpins structure, in which the amino- and carboxyl-terminal regions of the gp41 ectodomain are brought together. Synthetic peptides derived from these two regions (called N and C peptides, respectively) inhibit HIV-1 entry. In contrast to C peptides, which inhibit in the nanomolar range, N peptides are weak inhibitors with IC(50) values in the micromolar range. To test the hypothesis that the weak inhibition of N peptides results from their tendency to aggregate, we have constructed chimeric variants of the N-peptide region of gp41 in which soluble trimeric coiled coils are fused to portions of the gp41 N peptide. These molecules, which present the N peptide in a trimeric coiled-coil conformation, are remarkably more potent inhibitors than the N peptides themselves and likely target the carboxyl-terminal region of the gp41 ectodomain. The best inhibitors described here inhibit HIV-1 entry at nanomolar concentrations.

Mechanisms of viral membrane fusion and its inhibition.

Viral envelope glycoproteins promote viral infection by mediating the fusion of the viral membrane with the host-cell membrane. Structural and biochemical studies of two viral glycoproteins, influenza hemagglutinin and HIV-1 envelope protein, have led to a common model for viral entry. The fusion mechanism involves a transient conformational species that can be targeted by therapeutic strategies. This mechanism of infectivity is likely utilized by a wide variety of enveloped viruses for which similar therapeutic interventions should be possible.

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.

Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues.

Coiled coils consist of two or more alpha-helices wrapped around each other with a superhelical twist. The interfaces between helices of a coiled coil are formed by hydrophobic amino acid residues packed in a "knobs-into-holes" arrangement. Most naturally occurring coiled coils, however, also contain buried polar residues, as do the cores of the majority of naturally occurring globular proteins. Two common buried polar residues in both dimeric and trimeric coiled coils are asparagine and glutamine. In dimeric coiled coils, buried asparagine, but not glutamine, residues have been shown to confer specificity of oligomerization. We have placed a glutamine residue in the otherwise hydrophobic interior of a stable trimeric coiled coil, GCN4-pII, to study the effect of this buried polar residue in a trimeric coiled-coil environment. The resulting peptide, GCN4-pIQI, is a discrete, trimeric coiled coil with a lower stability than GCN4-pII. The crystal structure determined to 1.8 A shows that GCN4-pIQI is a trimeric coiled coil with a chloride ion coordinated by one buried glutamine residue from each monomer.

The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha.

The binding of oxidized low-density lipoprotein (Ox LDL) by monocyte-macrophages causes pleiotropic effects, including changes in gene expression, and is thought to represent an early event in atherogenesis. The integral membrane glycoprotein CD36 appears to play a physiological role in binding and uptake of Ox LDL by monocyte-macrophages, although the molecular events associated with CD36-Ox LDL interaction are unknown. To approach this issue, we used CD36 transfected Chinese hampster ovary (CHO) cells, exposed them to Ox LDL, and determined changes in the activity of the transcription factor NF-kappaB. We report here that Ox LDL enhanced DNA binding activity of nuclear extracts to an NF-kappaB sequence following activation of CD36-producing CHO cells with the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). This enhanced DNA binding activity was inhibited by coincubation of CD36 transfected cells with the human CD36-specific antibody OKM5. We also determined that activation of NF-kappaB DNA binding activity required an intact carboxyl-terminal cytoplasmic segment on CD36. Our results support the idea that human CD36 mediates signal transduction events in response to Ox LDL.