Kinetic Dependence to HIV-1 Entry Inhibition

Abstract

HIV-12 cellular invasion proceeds through fusion of viral and cellular membranes, a process mediated by the viral surface glycoprotein Env in response to binding cellular receptors (1, 2). Functional Env is composed of two noncovalently linked subunits, gp120 and gp41, arranged as a trimer of heterodimers on the virion surface. The interaction of gp120 with cellular CD4 and a chemokine receptor promotes structural rearrangements that ultimately lead gp41 to insert its N-terminal fusion peptide segment into the target cell membrane (Fig. 1A). Eventually, gp41 collapses into a stable, compact trimer-of-hairpins conformation essential for efficient membrane fusion (3).The core of the gp41 trimer-of-hairpins is a bundle of six ␣-helices formed by two hydrophobic heptad repeat sequences (HR1 and HR2) within each of the three gp41 ectodomains (4 -7). The N-terminal HR1 segments form a central, trimeric coiled coil. The C-terminal HR2 segments pack in an antiparallel manner into hydrophobic grooves on the exterior of the HR1 coiled coil. The antiparallel packing of HR1 and HR2 segments ensures that the fusion peptide and transmembrane region of gp41 are brought into close proximity (Fig. 1A). Since the fusion peptide is inserted within the target cell membrane and the transmembrane region is embedded within the viral membrane, formation of the gp41 trimer-of-hairpins promotes the juxtaposition of the two membranes (6). The similarity of the gp41 trimer-of-hairpins to the fusion-active conformations of glycoproteins from distantly related enveloped viruses suggests that this structure plays a critical role in promoting viral membrane fusion (see Ref. 3 and references therein).Peptides derived from the HR2 region of the gp41 ectodomain can disrupt formation of the gp41 trimer-of-hairpins, thereby inhibiting HIV-1 entry (4, 8, 9). Denoted as C-peptides, they work in a dominant negative fashion by binding the gp41 HR1 regions exposed in the extended, prehairpin intermediate conformation (4, 10 -13). Once bound, they prevent association of the HR1 and HR2 segments, thus blocking viral membrane fusion. That C-peptides bind an intermediate state in the fusion process is supported by the following observations: 1) C-peptides can precipitate gp41 only after Env binds cellular receptors (12, 13); 2) the extent of inhibition depends on C-peptide concentration present during membrane fusion and not on preincubation levels (12); 3) conditions that alter the rate of gp41-mediated membrane fusion influence the inhibitory potency of C-peptides (14 -16); and 4) at intermediate concentrations that block fusion pore formation, C-peptides can lock gp41 in a state that still permits lipid mixing (17). Despite the transient exposure of their binding site, some C-peptides possess potent (low nanomolar) inhibitory activity in vitro. One C-peptide, T20 (enfuvirtide), also demonstrates antiviral activity in vivo and has been approved by the FDA for treatment of HIV-1 infected individuals (18,19).In a complementary manner, agents that specific...

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates

“…More fundamentally, it remains to be seen whether a robust antibody response targeting the transient and sterically occluded prehairpin intermediate can be elicited. Recent studies suggest that access to this region during the viral fusion process may be limited by steric hindrance, which would be a significant barrier to the development of an effective vaccine (39,40). Our observation that neutralization potency and the quantity of D5-like antibodies are lower in rabbits than in guinea pigs may point to increased difficulty in eliciting protective neutralizing antibody titers in higher species, including primates.…”

Potent D-peptide inhibitors of HIV-1 entry

“…The trimer's binding to the pocket was too tight (low to mid pM) to measure accurately by SPR (the value reported in Table 1 is approximate and likely underestimates the trimer's true affinity). Interestingly, the trimer's potency against HXB2 did not improve as much as expected from its K D , suggesting that trimer potency against HXB2 may have reached a potency limit imposed by association kinetics, as has been reported for another entry inhibitor, 5-helix (34). This kinetic limitation is expected because the exposed N-trimer has an Ϸ10-to 20-min lifetime in the gp41 prehairpin intermediate (4)(5)(6), similar to the time required for binding of our peptides at mid to high pM concentrations.…”

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus

“…the HR1/HR2 bundle (8,16) although more recent results have demonstrated that this relationship may be complex (32,33). Using circular dichroism, the stability of the engineered HR2 peptides was determined in complex with the 51-aa HR1 target, T-865 (13).…”