Design of an engineered N‐terminal HIV‐1 gp41 trimer with enhanced stability and potency

Abstract

HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six-helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30-fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1-resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 Å crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six-helix bundle. These results provide an initial model of the pre-fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides.

Selection with a Peptide Fusion Inhibitor Corresponding to the First Heptad Repeat of HIV-1 gp41 Identifies Two Genetic Pathways Conferring Cross-Resistance to Peptide Fusion Inhibitors Corresponding to the First and Second Heptad Repeats (HR1 and HR2) of gp41

“…HR1 peptides additionally may interact with the HR1 of gp41 in a dominant negative mechanism to form a heterologous peptide-gp41 coiled coil that interferes with the endogenous coiled coil and prevents formation of the gp41 6HB (7,77,78). Since HR1 and HR2 peptides can target different sites and residues in gp41, HR1 peptides potentially represent different subclasses of fusion inhibitors with different resistance profiles.In studies aimed at understanding the mechanism of HR1 peptide inhibition and resistance, we (16) along with others (17,30) found that viruses resistant to HR1 peptide inhibitors are associated with the mutations in HR1 and HR2. Surprisingly, some of these initial reports also showed that these mutations conferred cross-resistance to HR2 peptide inhibitors (16) and, in some cases, increased in 6HB stability (16, 30).…”

“…In studies aimed at understanding the mechanism of HR1 peptide inhibition and resistance, we (16) along with others (17,30) found that viruses resistant to HR1 peptide inhibitors are associated with the mutations in HR1 and HR2. Surprisingly, some of these initial reports also showed that these mutations conferred cross-resistance to HR2 peptide inhibitors (16) and, in some cases, increased in 6HB stability (16, 30).…”

Peptides in the treatment of AIDS

“…Recently, single N-HR peptides were engineered to form soluble, stable trimeric coiled-coils, and their crystal structures were similar but not identical to the trimeric core found in the 6-HB [42]. The study demonstrated that N-peptides inhibit entry by binding C-HR peptides, that the predisposition of N-peptides to form trimeric coiled-coils may be more important for anti-viral activity than the Tm of the oligomer, and that binding of C-HR to N-HR may result in conformational changes.…”

Mechanism of resistance to S138A substituted enfuvirtide and its application to peptide design

“…Peptides corresponding to amino acid sequences of the gp41 carboxyl-terminal heptad repeat (C-HR) inhibit the HIV-1 fusion by acting as decoys and interfering with the formation of the six-helix bundle (Chan et al, 1998, Malashkevich et al, 1998). Although modified peptides such as SC34EK (Nishikawa et al, 2009), T-2635 (Dwyer et al, 2008), and D-peptides (Welch et al, 2007), and small molecules (Debnath et al, 1999) have been developed, T-20 (enfuvirtide) is the only fusion inhibitor approved for HIV therapy. It is a 36 amino acid peptide derived from the sequence of C-HR of gp41.…”