Figure 1.
Characteristics of C34 and C34-cholesterol.
(A) Schematic representation of HIV-1 gp41 main domains, depicting the relative position of the fusion peptide (FP), N-terminal heptad repeat domain (NHR), C-terminal heptad repeat domain (CHR) and transmembrane region (TM). The sequence of C34 was aligned with enfuvirtide, T-1249 and sifuvirtide, showing the pocket binding domain (PBD) and the lipid binding domain (LBD). Hydrophobic residues are represented in blue, non-charged polar residues in green, and charged polar residues in red. (B) Normalized fluorescence excitation (dashed line) and emission (solid line) spectra of 5 µM C34, C34-cholesterol or Trp in aqueous solution.
Figure 2.
Partition of the peptides to lipid vesicles.
Evaluation of Trp fluorescence variations of 5 µM C34 (A) or C34-cholesterol (B) upon titration with large unilamellar vesicles (LUV), performed by successive additions of POPC, POPC:Chol 2∶1, DPPC or HIV-like mixture LUV suspension. Dashed lines are fittings of eq. 1 to the experimental data.
Table 1.
Partition coefficients.
Figure 3.
Interaction of the fusion inhibitor peptides with lipid monolayers.
(A−C) Changes in surface pressure as a function of HIV-1 fusion inhibitor addition to pure POPC (A), POPC:Chol 2∶1 (B) or POPC:Chol:SM 1∶1:1 (C) monolayers. Dashed lines are fittings of eq. 2 to the experimental data. (D) Changes in surface pressure as a function of time after addition of HIV fusion inhibitors to achieve a final concentration of 31 nM on a POPC:Chol 2∶1 monolayer. All assays were carried out at 25°C, using an initial pressure of 21.5±0.5 mN/m. Each point is the average of at least triplicates of independent samples. Error bars represent the standard error of mean (SEM).
Table 2.
Dissociation constants, Kd, and ΔΠmax determined from surface pressure changes.
Figure 4.
Accessibility of the peptide to the aqueous medium.
Fluorescence quenching by acrylamide of C34 (A) and C34-cholesterol (B) in the presence (filled symbols) and absence (empty circles) of lipid vesicles (5 µM peptide and 3 mM total lipid). Lipid compositions tested were pure POPC, POPC:Chol 2∶1 and pure DPPC. Dashed lines are fittings of the Stern-Volmer equation (eq. 3) to the experimental data.
Figure 5.
Localization of C34-cholesterol in the bilayer.
(A–B) Stern-Volmer plots for the quenching of C34-cholesterol fluorescence by 5NS or 16NS in POPC (A) and POPC:Chol 2∶1 (B) LUV, using time-resolved fluorescence measurements. Each point is the average of three independent measures. The dashed lines are fittings of the Lehrer equation (eq. 4) to the experimental data, except for 16NS in POPC (eq. 3). (C) Depth of insertion of C34-cholesterol Trp residues in the membrane using SIMEXDA method [36], yielding an average location 16.8 Å away from the center of the bilayer for POPC and 18.0 Å for POPC:Chol 2∶1. Distributions’ half-width at half-height were 8.9 Å for POPC and 6.5 Å for POPC:Chol.
Figure 6.
Peptide interactions with di-8-ANEPPS labeled LUV and cell membranes.
(A–C) Differential spectra of di-8-ANEPPS bound to LUV (A) erythrocytes (B) and PBMC (C) membranes in the presence of C34-cholesterol and in its absence. Spectra were obtained by subtracting the excitation spectrum (normalized to the integrated areas) of labeled cells in the presence of peptide from the spectrum in the absence of the respective peptide. The shift to the red (decrease in dipole potential) is peptide concentration-dependent. For LUV, spectra traces represent C34-cholesterol 4 µM, in the presence of different lipid compositions: POPC, POPC:Chol 2∶1 and POPC:Chol 1∶1. For cells, spectra traces represent different C34-cholesterol concentrations: 0 µM, 0.25 µM, 1 µM and 5 µM. (D) Binding profiles of C34-cholesterol to LUV of POPC, POPC:Chol 2∶1 and POPC:Chol 1∶1, by plotting the di-8-ANEPPS excitation ratio, R (I455/I525, normalized to the initial value), as a function of the peptide concentration. DMSO, cholesterol and C34 (unconjugated) were also tested, as controls, and no significant changes on the dipole potential were observed (data not shown). (E–F) Binding profiles of C34-cholesterol, C34 and cholesterol to erythrocytes (E) and PBMC (F) cell membranes. Controls for DMSO (empty circles) and DMSO:ethanol (empty square) were also performed. Dashed curves represent the fitting to the single binding site equation (eq. 5). Affinity parameters for the cells are indicated in Table 3. Error bars represent SEM, with n = 5 for C34-cholesterol curves in cells and n = 3 for the control curves and LUV.
Table 3.
Antiviral activity and cell membrane interaction parameters compared of HIV-1 fusion inhibitors.
Figure 7.
Putative mode of action of HIV-1 fusion inhibitor C34-cholesterol.
In aqueous solution, the conjugate may form micro-aggregates when membranes are not present, due to its cholesterol moiety. The drug was demonstrated to partition to cell membranes and virus-like membranes. A preference towards cholesterol and SM-rich compositions was identified. These lipids are characteristic of the membrane microdomains designated as lipid rafts, which usually contain the receptors involved in HIV entry. C34-cholesterol anchors to the membrane via its cholesterol moiety and also, with a putative weaker binding, via its Trp (W)-rich N-terminal domain. In the context of HIV-1 gp41 engagement with the target cell, a confined space exits between the two membranes. Enrichment in DHSM in the viral membrane decreases the peptide partition, possibly shifting membrane partition equilibrium to the host cell membrane. The drug concentrated in the lipid raft environment may reach its target (gp41) more efficiently than through simple diffusion in aqueous solution. Moreover the anchoring promoted by the cholesterol at the C-terminus brings the peptide into contact in the correct orientation to compete with NHR binding site. This way, gp41 mediated fusion may be inhibited, blocking viral content entry into the cell.