Synthesis of Novel Mannoside Glycolipid Conjugates for Inhibition of

Article pubs.acs.org/bc

Synthesis of Novel Mannoside Glycolipid Conjugates for Inhibition of HIV‑1 Trans-Infection Laure Dehuyser,†,§ Evelyne Schaeffer,*,‡,§ Olivier Chaloin,‡ Christopher G. Mueller,‡ Rachid Baati,*,† and Alain Wagner† †

Laboratory of Functional Chemo Systems, CNRS-UdS UMR 7199, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67400 Illkirch, France ‡ Laboratory of Immunology and Therapeutic Chemistry, CNRS UPR 9021, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67000 Strasbourg, France S Supporting Information *

ABSTRACT: Mannose-binding lectins, such as dendritic cellspecific ICAM-3-grabbing non-integrin (DC-SIGN), are expressed at the surface of human dendritic cells (DCs) that capture and transmit human immunodeficiency virus type-1 (HIV-1) to CD4+ cells. With the goal of reducing viral trans-infection by targeting DC-SIGN, we have designed a new class of mannoside glycolipid conjugates. We report the synthesis of amphiphiles composed of a mannose head, a hydrophilic linker essential for solubility in aqueous media, and a lipid chain of variable length. These conjugates presented unusual properties based on a cooperation between the mannoside head and the lipid chain, which enhanced the affinity and decreased the need for multivalency. With an optimal lipid length, they exhibited strong binding affinity for DCSIGN (Kd in the micromolar range) as assessed by surface plasmon resonance. The most active molecules were branched trimannoside conjugates, able to inhibit the interaction of the HIV-1 envelope with DCs, and to drastically reduce trans-infection of HIV-1 mediated by DCs (IC50s in the low micromolar range). This new class of compounds may be of potential use for prevention of HIV-1 dissemination, and also of infection by other DC-SIGN-binding human pathogens.

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INTRODUCTION Since the beginning of the AIDS pandemia in the early 1980s, there are 32 antiretroviral drugs approved for therapy. They target different stages of the human immunodeficiency virus type-1 (HIV-1) life cycle, such as entry, reverse transcription, integration, and maturation.1 Their drawbacks are virus-induced drug resistance and drug-induced side effects. A complementary approach is now dedicated to the search of inhibitors able to prevent mucosal infection and transmission, since the epidemic disease has been largely maintained by mucosal transmission.2,3 After crossing the mucosal barrier, HIV-1 infects dendritic cells (DCs), macrophages, and T lymphocytes, residing underneath epithelial layers. DCs are one of the first cell types to be infected by HIV-1, mostly the R5 strain.4−7 In addition, DCs contribute to early-stage viral dissemination and transmission by their ability to capture, transport virions, and mediate infection of new target cells.8−10 This so-called trans-infection can occur by distinct pathways. The first mechanism implies the DC-specific intercellular adhesion molecule 3 [ICAM-3]grabbing nonintegrin (DC-SIGN), a C-type lectin highly expressed at the surface of DCs.11−13 DCs capture HIV-1 through DC-SIGN and transfer them to target CD4+ cells through cell−cell junctions.10 The vast majority of virions transmitted in trans originate from the cell plasma membrane rather than from intracellular vesicles.14 Other studies found © XXXX American Chemical Society

that DCs transmit HIV-1 through a DC-SIGN-independent exocytic pathway that involves HIV-associated exosomes.15 DC-SIGN is a type II membrane protein with a short aminoterminal cytoplasmic tail, a neck region, and a single C-terminus carbohydrate recognition domain (CRD). The extracellular CRD is a tetramer stabilized by an α-helical stalk, which specifically recognizes glycosylated proteins, and binds ligands bearing mannose and related sugars. The interaction of this domain with high-mannose structures of glycoproteins is multivalent and calcium-dependent.16,17 It interacts with envelope glycoproteins from distinct viruses such as HIV-1, Ebola, Hepatitis C, and Dengue, 18−22 but also with mycobacteria,23 fungi,24 and parasites.25 The HIV-1 envelope consists of the gp120 glycoprotein, exposed on the surface of the virion, and the transmembrane protein gp41. The highly glycosylated gp120 interacts with the mannose-binding domain of DC-SIGN at the surface of DCs.26−28 Previous studies aimed at preventing HIV transmission by targeting DC-SIGN have reported the synthesis of multivalent carbohydrate-containing molecules. Indeed, monovalent carbohydrates present weak millimolar binding constants for Received: November 29, 2011 Revised: June 27, 2012

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dx.doi.org/10.1021/bc200644d | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

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Surface Plasmon Resonance (SPR). SPR experiments were performed using a Biacore 3000. Immobilization of human DC-SIGN (Lys62-Ala404, from R&D Systems) on a CM5 sensor chip (GE-Healthcare, Uppsala, Sweden) was performed by injecting 70 μL of DC-SIGN (50 μg/mL in formate buffer, pH 4.3) onto the surface activated with N-ethylN′-dimethylaminopropyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS), which gave a signal of approximately 8000 RU, followed by 20 μL of ethanolamine hydrochloride, pH 8.5, to saturate the free activated sites of the matrix. Biosensor assays were performed with HEPES-buffered-saline (10 mM HEPES, 150 mM sodium chloride, pH 7.4) containing 0.005% surfactant P20 and 5 mM CaCl2, as running buffer. All binding experiments were carried out at 25 °C with a constant flow rate of 20 μL/min. The compounds dissolved in the running buffer (0.39 to 50 μM) were injected for 3 min, followed by a dissociation phase of 3 min. The sensor chip surface was regenerated after each experiment by injecting 10 μL of EDTA 0.5 M pH 8.0. A simple channel activated by EDC/NHS and deactivated with ethanolamine was used as control. The kinetic parameters were calculated using the BIAeval 4.1 software. Analysis was performed using the simple Langmuir binding model with separate ka/kd (kon/koff). The specific binding profiles were obtained after subtracting the response signal from the control channel and from blank-buffer injection. The fitting to each model was judged by the χ2 value and randomness of residue distribution compared to the theoretical model (Langmuir binding 1:1). Cell Culture. MAGI-CCR5 cells are HeLa-CD4-LTR-LacZ cells expressing both CXCR4 and CCR5 coreceptors (received from NIH AIDS Research and Reference Program, from Dr. Julie Overbaugh37). HEK293T cells were used for HIV-1 production. MCF-7 cells were used for viability tests. All cell lines were cultured at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with gentamycin and 10% (v/v) heat-inactivated fetal bovine serum (FBS). Generation of Human Monocyte-Derived Dendritic Cells. Elutriated human monocytes were obtained from the French Blood Bank (Etablissement França is du Sang, Strasbourg, France). To generate monocyte-derived dendritic cells, monocytes were cultured at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% FBS, gentamycin, 50 ng/mL of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF; ImmunoTools), and 10 ng/mL of recombinant human interleukin-4 (rhIL-4; ImmunoTools), with readdition of cytokines at day 3. Cells were harvested on day 5, and specific cell marker expression (CD1a, DC-SIGN) was characterized by flow cytometry on a FACS Calibur flow cytometer (Beckton-Dickinson) and analyzed with the CellQuest Pro software (BD Biosciences). Flow Cytometry. Expression of cell markers (CD1a, DCSIGN) was determined in FACS buffer (1% fetal bovine serum in PBS) at 4 °C for 20 min, using specific antibodies and their corresponding isotype controls (from BD-Pharmingen): CD1aallophycocyanin (HI149), CD209/DC-SIGN-PerCP-Cy5.5 (DCN46). Competition Assays between TriManC24 and HIV-1 gp120. Human DCs (1 × 105 in 100 μL) were aliquoted in a 96-well microtiter plate in RPMI 1640 medium (without FBS). To assess the binding of HIV-1 glycoprotein 120, FITCconjugated recombinant gp120 HIV-1 IIIB (ImmunoDiagnostics Inc., Woburn, MA, USA) was added to the cells (final

protein−carbohydrate recognition. It has been well recognized that the multivalent effect improves binding affinities by orders of magnitude. DC-SIGN binds to Man(9)GlcNAc(2) oligosaccharide 130-fold more tightly than mannose.17 Mannose dendritic polymers were found to interfere with the interaction between DC-SIGN and gp120 proteins.29,30 Two recent reports have described that multivalent mannoside polymers inhibit HIV transmission mediated by cell lines engineered to express DC-SIGN. First, gold mannoglyco-nanoparticles were shown to inhibit HIV trans-infection mediated by Raji-DCSIGN cells.31 Second, mannosyl glycodendritic structures, based on second and third generations of Boltorn hyperbranched dendritic polymers functionalized with mannose, inhibited HIV trans-infection mediated by THP-1-DC-SIGN cells.32 Distinct studies have described that the interaction between C-type lectins and natural lipomannans, a complex of fourdomain amphipathic molecules, is influenced by the presence of a lipid moiety. Indeed, the major cell-wall lipoglycans of Mycobacterium tuberculosis are high-molecular-weight mannosylated lipoarabinomannans (ManLAM) that strongly interact with DC-SIGN. Their lipid appendages, which are not directly linked to the mannose moieties, are thought to be involved in a supramolecular organization of the molecules by formation of micelles that increase ManLAM avidity for DC-SIGN.33,34 These data highlight the crucial role of lipids for creating a multivalent mannose presentation via structure formation into dynamic micelles. Therefore, with the goal of inhibiting HIV-1 trans-infection by blocking interactions with DC-SIGN, we decided to investigate the activity of low-molecular-weight mannosebased glycolipids. We designed a novel class of conjugates, formed by three building blocks that combine a polar mannose epitope moiety, a hydrophilic linker essential for water solubility, and a hydrophobic saturated lipid chain of variable length. Here, we describe their synthesis, their chemical and biological characteristics, namely, their affinity for DC-SIGN, their capacity to inhibit the binding of gp120 to DCs, and their ability to reduce HIV-1 trans-infection mediated by infected primary human immature DCs. Our findings revealed a branched trimannoside glycolipid conjugate able to drastically limit HIV-1 trans-infection.

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EXPERIMENTAL PROCEDURES

Compounds synthesis and characterization are described in Supporting Information. Critical Micelle Concentrations (CMC) and Dynamic Light Scattering (DLS). The CMC were measured using pyrene as an extrinsic fluorescent probe, as described.35 Briefly, a range of glycolipid samples were dissolved in water (1 mL), with concentrations ranging from 1.0 to 0.001 mg/mL. A stock solution of pyrene (1 mM) in dimethyl sulfoxide was added to each sample. Fluorescent emission of pyrene was measured at 25 °C using a Fluorolog spectrofluorometer (Jobin Yvon, Horiba). The intensities of fluorescence emission were determined at 374 nm (I374) and at 383 nm (I383), with an excitation at a 339 nm wavelength. The ratios I374/I383 were calculated and plotted against the glycolipid concentration. The CMC value was determined at the intersection of the two straight lines. The hydrodynamic diameters of micelles were measured in water by DLS measurements using a Zetasizer Nano ZS system (Malvern Instruments) as described.36 B



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Figure 1. Structure of mannoside glycolipid conjugates 1−8.

concentration of 5 μg/mL) and incubated for 45 min at 37 °C. For competition assays, cells were pretreated with either mannan (100 μg/mL final, corresponding to 620 μM of mannose units) or TriMan (100 μM) for 30 min at 37 °C, followed by incubation with gp120-FITC for 45 min at 37 °C. After washing with HBSS buffer (Lonza), expression of fluorescence intensity was analyzed on a FACS Calibur flow cytometer with the CellQuest Pro software. HIV-1 Production. Molecular clones of HIV-1 NL4−3 (X4 strain) and an NL4−3 R5-tropic version38 were transfected into HEK293T cells and viruses harvested from the supernatants 48 h later. All transfections were performed using calcium phosphate for precipitation of DNA. Viral stocks were normalized based on their p24 Gag content, measured in an enzyme-linked immunosorbent assay (ELISA) using the Innotest HIV antigen mAb kit (Innogenetics). HIV-1 Trans-Infection. Mannoside glycolipids (1.0 or 0.5 mM stock solution in water) were serially diluted in water. Human DCs (1 × 105 in 200 μL) were pretreated with different

concentrations of compounds, mannan (Sigma), or culture medium alone in RPMI 1640 medium containing 10% FBS for 45 min at 37 °C, prior to infection with HIV-1 (100 ng of p24Gag) for 3 h. Excess free virus and molecules were removed by three washes. DCs were then cocultured with MAGI-CCR5 cells (1 × 105) at 37 °C plated in a 48-well plate. After two days, viral infection of MAGI-CCR5 cells was quantitated following β-galactosidase assays and enumerating blue cells by light microscopy. The IC50s were determined by serial dilution of each compound. The trans-infection assays were repeated a minimum of three times, and the IC50s determined using Kaleidagraph.

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RESULTS AND DISCUSSION Synthesis of Monovalent Glycolipids Conjugates 1−4. Synthesis of the monosaccharide conjugates 1−4 (Figure 1) was achieved as depicted in Figure 2A. The linker between the mannose and the lipid chain was introduced for assuring

C



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Figure 2. Synthesis of linear and ramified mannoside glycolipids. (A) Synthesis of mannosides 1−4. Conditions: (i) TMSOTf, DCM, 0 °C to RT, overnight, 47%; (ii) PPh3, THF, H2O, RT, overnight, 81%; (iii) CH3COCl, THF, 0 °C to RT, overnight, 25%; (iv) NaOCH3, MeOH, RT, quantitative yield; (v) DCC, HOBT, THF, reflux, overnight, 9′, (14%); (vi) PPh3,THF, H2O, RT, overnight, 49%; (vii) DCC, HOBT, THF, reflux, overnight; lauric acid 17 (98%), stearic acid 18 (78%), pentacosanoic acid 19 (88%); (viii) NaOCH3, MeOH, RT, overnight, quantitative yield. (B) Synthesis of trivalent mannoside 7. Conditions: (i) Pentacosanoic acid 19, HOBt, DCC, THF, reflux, overnight, 77%; (ii) NaOH 4 N, THF/EtOH, RT, overnight, 94%; (iii) HOBt, HBTU, DIPEA, THF, reflux, overnight, 63%; (iv) NaOCH3, MeOH, RT, overnight, quantitative yield. (C) Synthesis of trivalent dimannoside 8. Conditions: (i) 9, TMSOTf, NIS, MS 4A, DCM, RT, overnight, 76%; (ii) K2CO3 (cat), MeOH, RT, overnight, 96%; (iii) 29, TMSOTf, NIS, MS 4A, DCM/Et2O, RT, overnight, 58%; (iv) PPh3, THF, H2O, RT, overnight, 61%; (v) 26, HOBt, HBTU, DIPEA, THF, reflux, overnight, 27%; (vi) K2CO3 (cat), MeOH, RT, overnight, quantitative yield; (vii) H2, Pd(OH)2/C, MeOH, RT, overnight, 97%.

the pivotal amine compound 13, which upon acetylation conditions and Zemplen final deprotection gave ManC1 1. Alternatively, amide coupling of 13 with PEG-carboxylic acid 9′ yielded sugar 15. After reduction of the azide function using Staudinger reduction to afford 16, the product was engaged in

solubility of the molecules in physiological media. The key intermediates PEG-azido linker 9′ and the glycosyl donor 11 were prepared from tetraethylene glycol and D-mannose 10, respectively.39,40 Briefly, glycosylation of 11 with alcohol 941 followed by the reduction of the azido function of 1242 afforded D



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dissociation equilibrium constant (KD = koff/kon) (Figure 3). As expected, mannan efficiently bound to the surface, while

amide bond formation with lauric 17, stearic 18, or pentacosanoic 19 acids to give, respectively, ManC11 (2), ManC17 (3), and ManC24 (4) in good chemical isolated yields (Supporting Information). Synthesis of the Multivalent Conjugate TriManC24 7. Based on previous studies showing that multiple mannose units, such as dendron-formed structures, are necessary for an optimal interaction with DC-SIGN, we synthesized multivalent compounds. To obtain 7 (Figure 2B), the synthesis of the key branched amine connector 24 was achieved.43 Treatment of 24 with pentacosanoic acid 19 and DCC yielded triester 25, which was saponified to tricarboxylic acid 26. Compound 26 was then reacted with sugar 13 under DCC/HOBt conditions to furnish trimannosylated product 27, which was deprotected to afford TriManC24 7. Synthesis of TriDiManC24 8. To further enhance the multivalent mannose presentation, we produced TriDiManC24 8 (Figure 2C). The glycosyl donor 29 was first synthesized.44−46 After glycosylation with alcohol 9 affording 30, the acetate function at C2 of 30 was selectively removed to yield the C2 free hydroxyl sugar acceptor 31. Subsequent glycosylation with glycosyl donor 29 gave the expected disaccharide 32. Selective reduction of the azido group using PPh3/H2O in standard conditions led to 33. Further coupling to the tricarboxylic acid connector 26 afforded the expected fully protected TriDiManC24 molecule. Removal of the acetates and global deprotection of the benzyl groups afforded TriDiManC24 8. Synthesis of Monovalent unsaturated Glycolipids Conjugates 5−6. To examine the importance of a saturated versus an unsaturated chain, we synthesized ManC24U 5, containing two triple insaturations, by amide bond formation between intermediate 16 and 10,12-pentacosadiynoic acid. As a control for the role of the sugar head, CellC24U 6 containing a cellobiose (or glucose−glucose) unit (Figure 1) was synthesized, as described in the Supporting Information. Properties of Mannoside Glycolipid Conjugates. All conjugates 1−8 could be solubilized in water by simple stirring, thanks to the hydrophilic linker. The saturated molecules exhibited no noticeable cytotoxicity on cells, as shown by viability assays on a human cell line and by dead cell staining of human DCs. The unsaturated compounds 5 and 6 were noncytotoxic up to 250 μM (Supporting Information). Micelles are dynamic systems that can spontaneously selfassemble above the critical micelle concentration (CMC), thus avoiding unnecessary construction steps to afford multivalent systems. Interestingly, when compounds were dissolved in water at 0.5 mg/mL, molecules ManC17 3, ManC24 4, and TriManC24 7 were able to auto-organize in micelles, at a CMC of, respectively, 112, 97, and 109 μM. The hydrodynamic diameter of these micelles was determined by dynamic light scattering and was, respectively, 17, 17, and 39 nm. Unsaturated ManC24U 5 had a lower CMC of 13 μM, and micelles had a hydrodynamic diameter of 11 nm. Molecules ManC1 1, ManC11 2, and TriDiManC24 8 were unable to form micelles. Affinities for DC-SIGN. Using surface plasmon resonance (SPR), we assessed the ability of our compounds to bind to the carbohydrate recognition domain (CRD) of DC-SIGN. A CM5 sensor chip was functionalized with the DC-SIGN CRD, and increasing concentrations of the various compounds, mannan as a positive control and octa(ethylene glycol) (OEG) as a negative control, were injected over the chip surface. By evaluation of the sensorgrams, we deduced the association (kon) and dissociation (koff) rate constants and the resulting

Figure 3. Direct binding by surface plasmon resonance (SPR) of mannoside glycolipid conjugates to DC-SIGN immobilized on a CM5 sensor chip. (A) Sensorgram of the binding of 7 to DC-SIGN (CRD) immobilized via amino groups. The binding response (in RU) was recorded as a function of time. (B) Kinetic parameters of interactions. kon, kinetic association rate; koff, kinetic dissociation rate; KD, equilibrium dissociation constant; Rmax (RU), maximal response expressed as resonance units; χ2, chi square value. 1:1 Langmuir binding model was used for all compounds, except for compound 3, for which steady-state affinity was used for calculation. With the control compound NHPD lacking mannose, no binding was detected at 100 μM. “N.b.”: No binding until 50 μM.

OEG was unable to bind. We found that ManC1 1 and ManC11 2 were unable to bind, ManC17 3 bound with a low affinity, while Man C24 4 exhibited an optimal affinity in the submicromolar range. These data showed that the affinity of the linear molecules for DC-SIGN correlated with the length of the lipid chain, suggesting that the lipid moiety of the moleculeat an optimal size of 24 carbon atomsenhanced the binding capacity of the mannose polar head in a cooperative manner. One mannose unit in ManC24 4 was sufficient for an effective interaction, with a KD in the submicromolar range. TriManC24 7 and TriDiManC24 8 also efficiently bound to DC-SIGN, with the KD in the low micromolar range, although they presented no improvement over ManC24 4. Similarly, ManC24U 5 containing an unsaturated lipid chain exhibited a KD in the low micromolar range, showing that these unsaturations did not improve the binding relative to a saturated chain. When the mannose head was replaced by a glucose unit (cellobiose), the binding was lost, which clearly revealed the importance of the mannose head. Similarly, when the sugar head was lacking, in NHPD composed of a linker conjugated to a 24-carbon lipid chain (see Figure 1), no binding was detected, which allowed us to rule out nonspecific binding. These results stressed the E



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importance of the mannose head, as well as of the 24-carbon saturated or unsaturated lipid chain. Altogether, these data demonstrated that glycolipid conjugates, formed by a C24-lipid chain covalently linked to one, three, or six mannose units, bind to the CRD of DC-SIGN with a good affinity. It is worth noting that ManC24 4, ManC24U 5, TriManC24 7, and TriDiManC24 8 interacted with DC-SIGN at concentrations below their CMC; the binding was effective below 10 μM, as visualized on the sensorgram Figure 3A. Thus, although molecules 4, 5, and 7 are able to form micelles, while 8 is unable to form micelles, they could all bind to DC-SIGN as single molecules, and micelle formation was not required. This contrasts with mycobacterial mannosylated lipoarabinomannans (ManLAM), in which terminal mannose residues and the lipid moiety are critical for binding to the pulmonary surfactant protein A (SP-A). In this case, lipids are required for an aggregate/micellar organization of the lipoglycan, since interaction with SP-A is lost in the presence of a low concentration of mild detergent (0.0005% of Polysorbate 20).47 In similar detergent conditions, binding of our mannosylatedC24 lipids to DC-SIGN was conserved, confirming that a process of supra-macromolecular structure formation is not required for binding to DC-SIGN. Binding Competition with HIV-1 gp120. We examined the ability of our compounds to inhibit the interaction of HIV-1 gp120 with DC-SIGN expressed at the surface of DCs. Immature DCs were prepared by differentiation of human monocytes. The expression levels of DC-SIGN were controlled by flow cytometry. As expected, results showed a high level of expression on DCs but no expression on monocytes, as indicated by the mean fluorescence index (MFI) (Figure 4A). When cells were incubated with fluorescently labeled gp120, we observed a strong fluorescence for DCs but not for monocytes, demonstrating more gp120 binding to DCs (Figure 4B). Some binding to monocytes was observed, likely owing to interaction with other cell surface proteins.48 We then performed competition assays by preincubating DCs with either compound TriManC24 7 or mannan followed by addition of fluorescently labeled gp120 (Figure 4C). Flow cytometry showed that 7 was able to compete with gp120FITC for the binding to DCs. Although inhibition was not complete, 100 μM of TriManC24 7 interfered better than mannan (620 μM of mannose units) with the binding of gp120 to DCs. Inhibition of HIV-1 trans-Infection Mediated by DCs. To investigate the ability of our compounds to inhibit HIV-1 trans-infection, we chose to infect DCs to best mimic in vivo conditions of viral capture by mucosal DCs. The DCs were mixed with the different compounds for 45 min at 37 °C, before exposure to HIV-1. After a 3 h incubation, the cells were extensively washed to remove unbound virions and mannosides and then cocultured with MAGI-CCR5 cells. This procedure allows precise quantification of HIV-1 replication in reporter MAGI-CCR5 cells by detection of β-galactosidase activity, since these cells express an integrated copy of the HIV long terminal repeat (LTR) fused to the β-galactosidase reporter gene. MAGI-CCR5 express the CD4 receptor and CCR5 and CXCR4 coreceptors, required for entry of, respectively, HIV1 R5 and X4. We first exposed DCs to the HIV-1 R5 strain, mostly involved in mucosal infections,7,49,50 in the presence of the linear glycolipids 1−6 (Figure 5A). While ManC1 1 and ManC11

Figure 4. TriManC24 7 competes with HIV gp120 binding to dendritic cells (DCs). (A) Cell surface expression of DC-SIGN on DCs and monocytes, analyzed by flow cytometry. Filled and gray histograms represent, respectively, antigen labeling and isotype controls. Values of mean fluorescence intensity (MFI) for antigen labeling are indicated. (B) Interaction of gp120-FITC with monocytes and DCs, after incubation for 45 min at 37 °C (filled histograms). Gray histograms represent untreated cells, in the presence of IgG1-FITC control. (C) DCs were exposed to TriManC24 7 (100 μM) or mannan (100 mg/mL or 620 mM of mannose units) for 30 min or left untreated, followed by incubation with gp120-FITC for 45 min at 37 °C. Flow cytometry open and filled histograms correspond to cells binding gp120-FITC, respectively, in the absence and presence of pretreatment. Results are from one representative experiment repeated three times.

2 did not present better inhibitory activity than mannan, ManC17 3, ManC24 4, and ManC24U 5 clearly reduced transinfection in a dose-dependent manner, more efficiently than mannan (0.6 and 1.5 mM of mannose units). This inhibition was in the high micromolar range with an IC50 of 120 μM. Interestingly, CellC24U 6 was unable to affect trans-infection, which showed the importance of the mannose head in the inhibition process. Dose−response experiments were then performed to compare the inhibitory capacity of the linear ManC24 4 and branched TriManC24 7 and TriDiManC24 8. When compared with ManC24 4, TriManC24 7 displayed a more potent inhibitory activity in the submicromolar range (IC50 of 0.5 μM). At the highest concentrations, trans-infection was drastically reduced, down to a residual level of 20% (Figure 5B). TriDiManC24 8 exhibited a powerful inhibitory activity in the nanomolar range (IC50 of 38 nM), although, surprisingly, infection leveled off at 40% (Figure 5C). The residual level of trans-infection may be explained by the previously reported distinct mechanisms of viral transmission, besides the transfer mediated by DC-SIGN. We further examined the effect of TriManC24 7 on the HIV-1 X4 strain (Figure 5D). Similar to the effect on the R5 strain, it also inhibited HIV-1 X4 trans-infection down to a residual level of 15%, although with a higher IC50 (7 μM). These findings demonstrate that TriManC24 7 is an active mannoside glycolipid F



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Figure 5. Inhibition of HIV-1 trans-infection by glycolipid conjugates. DCs were pretreated with the indicated compounds for 30 min at 37 °C, and then infected with HIV-1. After extensive washes, DCs were cocultured with MAGI-CCR5 cells for 2 days. Viral trans-infection was quantified. Values were normalized relative to 100% infection obtained with medium alone. Values are means ± SE from data obtained at least from three independent experiments. (A) Effect of the length of the carbon chain and of the sugar (either mannose or glucose). Mannan was included as a positive control. (B) Dose−response results for HIV-1 R5 exposed to mono- and trimannosides. (C) HIV-1 R5 exposed to tri- and tri-bismannosides. (D) HIV-1 X4 exposed to TriManC24 7.

conjugate to reduce trans-infection of HIV-1 R5 and X4 mediated by DCs.

other while bound to the same or adjacent DC-SIGN molecules simulating a bivalent ligand that bridges binding sites. Such a chelating binding has been shown before to strongly increase binding affinity.51,52 The precise role of the lipid chain in the molecular interactions between the compounds and DC-SIGN remains the subject of further studies. Our findings reveal that, thanks to this original cooperation, such conjugates are able to target DC-SIGN and inhibit HIV-1 trans-infection without the need for synthesis of previously reported complex mannosides structures.31,32 Nevertheless, it is clear that the multivalent presentation of 6 and 3 mannoses helped improve the transinfection ability. The most likely hypothesis accounting for inhibition of HIV1 trans-infection relies on the shield model: interaction of the compounds with DC-SIGN at the DC cell membrane prevents the attachment of HIV-1 to this receptor. In addition, partial down-regulation of DC-SIGN from the cell surface may also enhance the protective effect. One could also hypothesize a direct effect of the compounds on the virus particle. The molecule may disrupt the viral membrane by insertion of its lipid moiety, or interact via its mannose units with gp120 at the surface of the virus. Clearly, further studies are needed to shed more light on the precise inhibitory mechanism. Finally, the interest of this novel class of inhibitory molecules also lies in their potential ability to target other viruses and pathogens that use DC-SIGN or other C-type lectins for entry and infection.18−25

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CONCLUSIONS Our studies revealed the unexpected properties of a new class of glycolipid conjugates. These compounds are formed by three building blocksa mannose head, a linker, and a C24 lipid chainand are able to efficiently target DC-SIGN. Surprisingly, our data showed that a scaffold offering a multivalent mannose presentation, to mimic the high mannose structure occurring in glycoproteins, was not required for obtaining high binding affinity for DC-SIGN. Our SPR results demonstrated that the affinity of the mannose moiety for the extracellular domain of DC-SIGN could be greatly increased by coupling the mannose unit to a lipid chain of at least 17 carbons. The activities of these conjugatestheir affinity for DCSIGN, their ability to compete for gp120 binding, and their inhibition of HIV-1 trans-infectionare based on the cooperation between the lipid chain and the linked mannose moiety. The lipid chain was originally synthesized for molecular structure formation into self-organized micelles, and thus afford a multivalent mannose presentation. Interestingly, we discovered that such a structure formation was not required, neither for binding to DC-SIGN nor for inhibiting HIV trans-infection, as revealed by ManC24 4. Indeed, TriManC24 7 and TriDiManC24 8 inhibited viral trans-infection at an IC50 of, respectively, 500 and 38 nM, well below their CMC, showing that they possess the property of being active not only as micelles, but also as individual molecules. It is likely that, in addition to the interaction of the mannose unit with the CRD, the lipid chain interacts with distinct amino acids of DC-SIGN, thus optimizing the binding. It could also be that the lipid chains of two molecules make hydrophobic interactions with each

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ASSOCIATED CONTENT

* Supporting Information S

Full synthetic and characterization details of the compounds including NMR spectra, and cell viability assays are provided. G



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This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: e.schaeff[email protected] (E.S.); baati@ bioorga.u-strasbg.fr (R.B.). Author Contributions §

These authors contributed equally to this work.

Funding

This work was supported by the “Centre National de la Recherche Scientifique” (CNRS). The University of Strasbourg is acknowledged for a research grant to L.D. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to P. Pévet and S. Reibel-Foisset for the use of the A3/L3 platform of the Institut Fédératif de Recherches (IFR37), Strasbourg. We thank the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for MAGI-CCR5 cells.

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ABBREVIATIONS DC-SIGN, dendritic cell-specific ICAM-3-grabbing nonintegrin; DCs, dendritic cells; HIV-1, human immunodeficiency virus type-1; CRD, carbohydrate recognition domain; CMC, critical micelle concentration; SPR, surface plasmon resonance

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REFERENCES

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