Palmitic Acid Is a Novel CD4 Fusion Inhibitor That Blocks HIV Entry and Infection

Abstract

The high rate of HIV-1 mutation and the frequent sexual transmission highlight the need for novel therapeutic modalities with broad activity against both CXCR4 (X4) and CCR5 (R5)-tropic viruses. We investigated a large number of natural products, and from Sargassum fusiforme we isolated and identified palmitic acid (PA) as a natural small bioactive molecule with activity against HIV-1 infection. Treatment with 100 microM PA inhibited both X4 and R5 independent infection in the T cell line up to 70%. Treatment with 22 microM PA inhibited X4 infection in primary peripheral blood lymphocytes (PBL) up to 95% and 100 microM PA inhibited R5 infection in primary macrophages by over 90%. Inhibition of infection was concentration dependent, and cell viability for all treatments tested remained above 80%, similar to treatment with 10(-6)M nucleoside analogue 2', 3'-dideoxycytidine (ddC). Micromolar PA concentrations also inhibited cell-to-cell fusion and specific virus-to-cell fusion up to 62%. PA treatment did not result in internalization of the cell surface CD4 receptor or lipid raft disruption, and it did not inhibit intracellular virus replication. PA directly inhibited gp120-CD4 complex formation in a dose-dependent manner. We used fluorescence spectroscopy to determine that PA binds to the CD4 receptor with K(d) approximately 1.5 +/- 0.2 microM, and we used one-dimensional saturation transfer difference NMR (STD-NMR) to determined that the PA binding epitope for CD4 consists of the hydrophobic methyl and methelene groups located away from the PA carboxyl terminal, which blocks efficient gp120-CD4 attachment. These findings introduce a novel class of antiviral compound that binds directly to the CD4 receptor, blocking HIV-1 entry and infection. Understanding the structure-affinity relationship (SAR) between PA and CD4 should lead to the development of PA analogs with greater potency against HIV-1 entry.

FIG. 1.

Inhibition of X4 and R5-tropic HIV-1 infection and cell viability. (A) Flow cytometry analyses of GHOST X4/R5 GFP-expressing cells that were treated overnight with increasing micromolar concentrations of PA indicated on top of each box, washed, and infected for 1.5 h at 0.3 MOI in replicates (n = 3), with NL4-3 (X4 infection) or with 81A (R5 infection). The percentage of infected cells is indicated inside each box, and % inhibition is indicated below each box. Uninfected and untreated control is superimposed over each histogram by a dotted line. (B) Human PBL were treated overnight with increasing micromolar concentrations of PA or 10⁻⁶ M ddC, washed, and infected with NL4-3 at 0.1 MOI for 2 h in the absence of each treatment, washed three times, and returned to culture with each respective treatment for the duration of the experiment; (left panel) p24 pg/ml antigen production in cell-free supernatants at the peak of infection on day 6; (middle panel) calculated % inhibition; and (right panel) % viability. (C) Human macrophages (Mφ) were treated overnight with increasing micromolar concentrations of PA or with 10⁻6 M ddC, infected with HIV-1 R5 primary isolate ADA at 0.2 MOI for 18 h in the presence of each treatment, washed three times, and returned to culture with each respective treatment for the duration of the experiment; (left panel) p24 pg/ml antigen production in cell-free supernatants at the peak of infection on day 10; (middle panel) calculated % inhibition; and (right panel) % viability. Bars indicate ± SD, representative of three experiments.

FIG. 2.

Inhibition of cell-to-cell and virus-to-cell fusion. (A) GHOST GFP-expressing cells were pretreated overnight with 0, 20, or 40 μM PA and cocultivated at a 1:1 ratio with NL4-3-infected CEM cells for 24 h. Mixed cell cultures were examined by flow cytometry and percent of GFP-expressing fused cells is indicated inside each panel. Mock cells were cocultivated with uninfected CEM cells and analyzed for background GFP expression. Percent PA-mediated inhibition is indicated on top of each panel. (B) SupT1 cells were treated overnight as indicated on the top of each box, washed, loaded with CCF2/AM dye, and then infected with fusion competent BlaM-Vpr NL4-3 in the absence of treatment. Virus–cell fusion was measured by cleavage of CCF2 dye. Cells were analyzed by multiparameter flow cytometry using a violet laser for excitation of CCF, gated from 10,000 cells. Percent virus-to-cell-fused cells (FUS) and % inhibition (INH) are indicated inside each box. Results are representative of three independent experiments.

FIG. 3.

Analysis of cell surface CD4 receptor and lipid rafts distribution. (A) Sup-T1 cells were treated overnight with increasing concentrations of PA, or for 5 h with 10 ng/ml PMA that is a positive control for CD4 internalization. All systems were washed and surface labeled with PE-conjugated anti-CD4 antibody, results of which were superimposed over negative control (gray) unstained cells. Cells were analyzed on a BD LSR II flow cytometer. Internalization of cell surface CD4 was measured by the shift in the mean fluorescent intensity, and percent internalized (Int) and percent surface (Sur) CD4 receptor are marked inside each box. Representative of two independent experiments. (B) Lipid rafts of 1G5 T cells that were labeled with 1 μg/ml CTB-HRP. Labeled cells were then incubated for 1 h in media containing 20 μM palmitic acid (PA), 20 μM myristic acid (MA), or no fatty acid (No Treatment). Cells were then lysed in cold 0.1% Triton X-100 and lipid rafts were isolated by sucrose density gradient centrifugation. Five gradient fractions were collected and assayed for HRP activity. The CTB-HRP distribution is reported as a percent of total activity. Lipid rafts are expected to be present in fractions 1 and 2, which contain the 5%/35% sucrose interface. Shown is the average CTB-HRP distribution for three independent experiments. Bars indicate ± 1 SD.

FIG. 4.

Palmitic acid inhibits gp120-CD4 complex formation and infection with CD4-dependent envelope. (A) Inhibition of gp120-CD4 complex formation was investigated by gp120 capture ELISA. Envelope gp120 (IIIB) protein was captured on 96-well plates, washed, and incubated in the presence of CD4-biotin alone or in the presence of increasing concentrations of PA, as indicated. Streptavidin-HRP was added, and then developed by the addition of a chemiluminescent substrate, o-phenylenediamine dihydrochloride (OPD). Reaction was stopped by adding 1 N HCl and read at 490 nm. Percent inhibition of the complex formation was calculated from untreated (0 μM PA) that was taken as 0% inhibition. (B) CEM cells were treated overnight with 10 or 20 μM PA, then washed and infected in the absence of treatment with infectious HIV-Env⁻Luc⁺ virus pseudotyped with either CD4-independent VSV-G or with CD4-dependent HXB2 envelope, to yield single round viral replication. Infection was quantified by luciferase expression 48 h after infection and reported as % inhibition of infection without treatment. Bars indicate ± 1 SD, representative of three independent experiments.

FIG. 5.

In vitro binding experiments and analysis of sCD4 with PA. (A) Homonuclear NMR spectrum of free 100 μM PA in the NMR buffer (10 mM KPO₄ buffer, pH 7.0, 20% d₆-DMSO, 80% D₂O). Thirteen PA methylene groups located away from the PA carboxyl terminal resonate at 1.5 ppm. PA methylene groups located close to the PA carboxyl terminal resonate at 2.27 ppm and 1.57 ppm, respectively. (B) STD-NMR signal of PA bound to sCD4. The increase of the methylene STD-NMR signal of PA at 1.5 ppm is observed with the increase of the PA concentration in the sample of 14 μM sCD4 dissolved in the NMR buffer: (1) no PA, (2) molar ratio of sCD4 to PA is 1: 0.1, (3) 1:0.6, (4) 1:0.8, (5) 1:1.2, (6) 1:2, (7) 1:3, (8) 1:5, (9) 1:7, and (10) 1:10. The nonzero STD-NMR signal of PA indicates that PA directly binds to sCD4. PA methelene groups –(CH₂)₁₃– that show a large STD-NMR signal constitute the binding epitope of PA for CD4. Peaks from the PA methylene groups located close to the PA carboxyl terminal that are not part of the binding epitope are suppressed in the STD-NMR spectrum. (C) Fractional STD effect of the –(CH₂)₁₃– signal at a given PA concentration. The gradual decrease of the STD effect indicates that the PA–sCD4 complex is specific. (D) Fluorescence titration experiment of sCD4 with increasing concentration of PA. This experiment was used to estimate the binding affinity of PA for sCD4. Tryptophan fluorescence was measured using an excitation wavelength of 280 nm. An increase of PA causes a red shift of 2 nm and quenching of the tryptophan fluorescence of sCD4. Inset: Binding isotherm of the normalized sCD4 tryptophan fluorescence with increasing concentration of PA at the emission wavelength of 350 nm. Curve fitting (OriginLab) using a single site binding isotherm approximation resulted in the best value for K_d to be 1.5 ± 0.2 μM.