Creating an Artificial Tail Anchor as a Novel Strategy To Enhance the Potency of Peptide-Based HIV Fusion Inhibitors

Abstract

20 (enfuvirtide) and other peptides derived from the human immunodeficiency virus type 1 (HIV-1) gp41 C-terminal heptad repeat (CHR) region inhibit HIV fusion by binding to the hydrophobic grooves on the N-terminal heptad repeat (NHR) trimer and blocking six-helix-bundle (6-HB) formation. Several strategies focusing on the binding grooves of the NHR trimer have been adopted to increase the antiviral activity of the CHR peptides. Here, we developed a novel and simple strategy to greatly enhance the potency of the existing peptide-based HIV fusion inhibitors. First, we identified a shallow pocket adjacent to the groove in the N-terminal region of NHR trimer as a new drug target, and then we designed several short artificial peptides to fit this target. After the addition of IDL (Ile-Asp-Leu) to the C terminus of CHR peptide WQ or MT-WQ, the conjugated peptides, WQ-IDL and MT-WQ-IDL, showed much more potent activities than WQ and T20, respectively, in inhibiting HIV-1 IIIB infection. WQ-IDL and MT-WQ-IDL were also more effective than WQ in blocking HIV-1 Env-mediated membrane fusion and had higher levels of binding affinity with NHR peptide N46. We solved the crystal structure of the 6-HB formed by MT-WQ-IDL and N46 and found that, besides the N-terminal MT hook tail, the IDL tail anchor of MT-WQ-IDL also binds with the shallow hydrophobic pocket outside the groove of the NHR trimer, resulting in enhanced inhibition of HIV-1 fusion with the target cell. It is expected that this novel approach can be widely used to improve the potency of peptidic fusion inhibitors against other enveloped viruses with class I fusion proteins.IMPORTANCE The hydrophobic groove of the human immunodeficiency virus type 1 (HIV-1) gp41 NHR trimer has been known as the classic drug target to develop fusion inhibitors derived from the gp41 CHR. Here, we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced, by 77-fold, making it much more potent than T20 (enfuvirtide) and suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR peptide fusion inhibitors. This new approach of identifying a potential binding pocket outside the traditional target and creating an artificial tail anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).KEYWORDS HIV, gp41, fusion inhibitor, six-helix bundle, peptide

Adding an Artificial Tail—Anchor to a Peptide-Based HIV-1 Fusion Inhibitor for Improvement of Its Potency and Resistance Profile

“…However, HIV-1 with E49K mutation in the gp41 NHR became resistant to HP23 [ 24 ]. In our previous study, we identified a shallow pocket (N-pocket) in the N-terminal region of the NHR domain ( Figure 1 b) and found that addition of an IDL-anchor to the C-terminus of a CHR-peptide could significantly improve the peptide’s anti-HIV-1 potency and resistance profile ( Figure 1 c) [ 27 ]. Therefore, we proposed that the addition of an IDL-anchor to the C-terminus of HP23 could result in more stable binding of this peptide with the groove on the NHR-trimer since its N- and C-terminal anchors could bind to the C- and N-pockets on the NHR-trimer, respectively ( Figure 1 c).…”

“…Our previous study has shown that addition of an MT anchor and an IDL anchor to the N- and C-termini of the peptide WQ, respectively, results in generation of a new peptide, designated MT-WQ-IDL, with significantly improved inhibitory activity against HIV-1 Bal infection (IC 50 = 0.6 nM) [ 27 ]. It was reported that addition of a glutamic (E) residue to the N-terminus of a CHR-peptide with MT-hook could stabilize the MT hook structure [ 21 ].…”

“…We thus expected that HP23-E6-IDL, which contains the EMT-anchor, might have more potent anti-HIV-1 Bal infection than MT-WQ-IDL. Interestingly, we found that although HP23-E6-IDL had equal potency against HIV-1 Bal infection (IC 50 = 0.6 nM) as that of MT-WQ-IDL, HP23-E6-IDL was generally more effective than MT-WQ-IDL against T20- and T2635-resistant strains [ 27 ]. These results suggest that addition of the EMT-anchor to a CHR-peptide is more favorable than addition of MT-anchor for designing CHR-peptide with improved antiviral activity against HIV-1 mutants resistant to CHR-peptides.…”

“…We have previously demonstrated that addition of an IDL (Ile-Asp-Leu) anchor to the C-terminus of a CHR peptide could improve the peptide’s anti-HIV-1 activity [ 27 ]. Here, we modified HP23 by extending its C-terminal sequence using six residues (E6) and adding IDL to the C-terminus of E6, which is expected to bind to the shallow pocket in the N-terminal region of the gp41 NHR-trimer.…”

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

“…The diffraction data sets for SC29EK/N36 were collected at beamline BL-19U1 at the Shanghai Synchrotron Radiation Facility, China. The phasing problem was solved by the molecular replacement method using the software CCP4i with a crystal structure of HIV gp41 core (PDB accession number 5H0N) as a searching model (45,46). The final model was manually adjusted in COOT based on the electron density map and refined with the program PHENIX (47,48).…”