Novel Recombinant Engineered gp41 N-terminal Heptad Repeat Trimers and Their Potential as Anti-HIV-1 Therapeutics or Microbicides

Abstract

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved ␣-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

“…Potent HIV inhibitors have been designed by sequestering the NHR peptide into a nonaggregating trimeric coiled-coil conformation, which should be able to effectively bind the CHR helix and interfere with six-helix bundle formation. Several design approaches of this type include covalent stabilization of the trimer by disulfide bridges (13)(14)(15) and fusion of NHR segments to trimerization domains (16)(17)(18). Also targeting the CHR region, a protein denoted 5-Helix was engineered by connecting five of six helices that make up the core of the gp41 postfusion structure using short peptide linkers (19).…”

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

“…We found that these mutants were highly resistant to C34, CP32M, and ADS-J1, but relatively sensitive to T20, suggesting that ADS-J1, C34, CP32M and T2635 share the same target: the pocket region of the HIV-1 gp41 [88–90]. Subsequently, we constructed a gp41 NHR-trimer by conjugating the NHR-peptide N36 with a trimerization motif, foldon (Fd), termed N36Fd, as previously described [91], and several N36Fd mutants, including N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd. We found that the binding affinity of the mutant N36Fd trimers to ADS-J1 was lower than that of the wild-type N36Fd trimer [88], further confirming that the gp41 pocket region is the main target of ADS-J1.…”

“…Therefore, establishing an HTS assay using the transiently exposed NHR coiled coil in solution is very challenging. To address this challenge, researchers have employed several strategies, including the design of 5-Helix protein, which contains 5 of the 6 helices that constitute the gp41 trimer-of-hairpins linked into a single polypeptide [100], the addition of a soluble trimeric motif, GCN4 or foldon to a NHR peptide, in order to stabilize the coiled coil [91, 101, 102], and the covalent linking of the peptides in the NHR-trimeric coiled coil [103]. …”

Targeting HIV-1 gp41-induced Fusion and Pathogenesis for Anti-viral Therapy

“…[98, 99] Another recombinant inhibitor was constructed using peptides derived from the N-terminal heptad repeat. N-terminal peptides are usually not considered to be good candidates because of their propensity for aggregation instead of formation of discrete coiled-coil trimers [100]. The novel protein is made up of N-peptides that have been fused to the T4 fibritin trimerization domain, Foldon.…”

“…The novel protein is made up of N-peptides that have been fused to the T4 fibritin trimerization domain, Foldon. This protein was able to form a stable six-helix bundle with the addition of C-peptide and showed potent inhibition against a broad spectrum of HIV strains in vitro [100]. …”