Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition

Abstract

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

Keywords: HIV-1; T20; fusion inhibitor; gp41; lipopeptide.

FIG 1

Schematic illustration of HIV-1 gp41 protein and its NHR- and CHR-derived peptides. The gp41 numbering of HIV-1_HXB2 was used. TM, transmembrane domain. The positions and sequences corresponding to the T20-resistant site and pocket-forming site in the NHR are marked in blue. The positions and sequences corresponding to the M-T hook structure, PBD, and tryptophan-rich motif in the CHR are marked in green, red, and purple, respectively. “C16” in inhibitors represents a fatty acid group.

FIG 2

Biophysical properties of T20-based inhibitors determined by CD spectroscopy. The α-helicity (A) and thermostability (B) of T20- and T20-TRM-based 6-HBs, the α-helicity (C) and thermostability (D) of LP-40- and DP-C16-based 6-HBs, and the α-helicity (E) and thermostability (F) of LP-40 with linker-based 6-HBs were determined, with the final concentration of each peptide in PBS at 10 μM. The α-helicity and T_m values are shown in parentheses. NA, not applicable for calculation. The experiments were repeated at least two times, and representative data are shown.

FIG 3

Interaction of LP-40 and control peptides with an NHR-derived peptide. (A to C) The thermodynamic profiles of the molecular interactions between T20 (A), T20-TRM (B), or LP-40 (C) and N39 were determined using ITC technology. The titration traces are shown at the top, and the binding affinities when the N39 solution was injected into an T20, T20-TRM, or LP-40 solution are shown at the bottom. (D) Visualization of the binding of LP-40 and control peptides to the NHR peptide N39 or N36 by native PAGE; each of the peptides was used at a final concentration of 40 μM. (E) Dose-dependent binding of T20 and LP-40 (40 μM) to N39 (0, 20, and 40 μM).

FIG 4

Binding models of membrane-anchoring lipopeptides. Both classes of lipopeptide inhibitors can bind to the extended PHI during the early stage of viral membrane fusion. LP-11 mainly targets the pocket site located at the extreme C terminus of the NHR helices, and thus, it needs a long linker to reach the position (binding mode I). LP-40 targets the membrane-proximal NHR site, and its anchoring via a lipid molecule does not affect its binding (binding mode II). Red represents peptide sequences, purple represents fatty acid molecules, and the black line represents a PEG 8 linker.

FIG 5

Synergistic anti-HIV effects of LP-40 and LP-11. The synergistic inhibition by inhibitors of diverse HIV-1 pseudoviruses was determined by single-cycle infection assay. Peptides were tested individually and in combination at a fixed molar ratio achieving equivalent IC₅₀s. LP-40/LP-11 ratios, 3:1 (A), 1:2 (B), and 2:1 (C). Each sample was tested in triplicate, and the experiment was repeated 3 times. An NL4-3 pseudovirus with a D36G mutation in gp41 was used. The error bars indicate standard deviations (SD).

FIG 6

Synergistic anti-HIV effects of unconjugated peptide inhibitors. The synergistic inhibition by inhibitors of HIV-1 pseudoviruses was determined by single-cycle infection assay. Peptides were tested individually and in combination at a fixed molar ratio achieving equivalent IC₅₀s. T20/HP23 ratios, 5:1 (A), 1:1 (B), and 1:2 (C); T20/T1249 ratios, 3:1 (D), 1:1 (E), and 3:1 (F). Each sample was tested in triplicate, and the experiment was repeated 3 times. The error bars indicate SD.

FIG 7

Intrahelical hydrogen bonds of LP-40 in the 6-HB structure. (A) Ribbon model of 6-HB structure formed by the N44/LP-40 complex (PyMol). The N44 trimer is colored gray. LP-40 inhibitors are colored green. Residues related to hydrogen bonds on LP-40 are shown as stick models colored green with labels. The C terminus of LP-40 and the N terminus of N44 are labeled. (B) Amino acid sequence of LP-40. The glutamine residues are colored red, the glutamic acid residues are blue, and the asparagine residue is green. The solid black lines indicate hydrogen bonds formed between residues on LP-40.

FIG 8

Interhelical salt bridges and hydrogen bonds in the 6-HB structure of N44/LP-40. (A) Portion of a ribbon model of the 6-HB structure formed by N44/LP-40 (PyMol). The N44 trimer is colored gray. Residues related to hydrogen bonds and salt bridges on the N44 trimer are shown as stick models colored gray with labels. The LP-40 inhibitors are colored green. Residues related to hydrogen bonds and salt bridges on LP-40 are shown as stick models colored green with labels. The dashed lines indicate salt bridges and hydrogen bonds between residues on the N44 trimer and the inhibitor. The N terminus of N44 and the C terminus of LP-40 are labeled. (B) View of the crystal structure in panel A rotated 90° to the left. The same representation and color scheme as in the crystal structure in panel A are used. (C) A single LP-40 interacting with two NHR helices shown in a sequence map. The positively charged residues related to salt bridges are red, and the negatively charged residues related to salt bridges are green. The dashed black lines indicate salt bridges and hydrogen bonds between the N44 trimer and LP-40. The C and N termini of the sequences are labeled.

FIG 9

Interactions of N and C termini of LP-40 in the 6-HB crystal structure. Hydrophobic interactions of LP-40 are presented horizontally in a ribbon model (PyMol). The N44 trimer is colored gray. Residues related to hydrogen bonds are shown as stick models in gray. Hydrophobic amino acids related to hydrophobic interaction on the N44 trimer are shown as stick models and colored in corresponding colors. LP-40 inhibitors are green. Hydrophobic amino acids related to hydrophobic interaction on LP-40 are shown as stick models and colored in corresponding colors. The N and C termini of LP-40 and the N terminus of N44 are labeled.