HIV and SIV Envelope Glycoproteins Interact with Glycolipids and Lipids.

The present study demonstrates that, in addition to interacting with galactosylceramide (GalCer), HIV-1, HIV-2, and SIV envelope glycoproteins are able to interact with glucosylceramide (GlcCer), lactosylceramide (LacCer), and ceramide. These interactions were characterized by using three complementary approaches based on molecular binding and physicochemical assays. The binding assays showed that iodinated radiolabeled HIV-1 and HIV-2 glycoproteins (125I-gp) interact physically with GalCer, GlcCer, LacCer, and ceramide previously separated by thin layer chromatography (TLC) or directly coated on a flexible 96-well plate. These interactions are specific as demonstrated, on the one hand, by the dose-dependent inhibition in the presence of various dilutions of immune, but not non-immune, sera, and, on the other hand, by the absence of interaction of these glycolipids/lipids with 125I-IgG used as an unrelated control protein. These interactions were further confirmed in a physicochemical assay, based on the capacity of these glycolipids for insertion in a pre-established monomolecular film, as a model of the cell membrane, with each glycolipid/lipid. The addition of HIV envelope glycoproteins, but not ovomucoid protein used as a negative control, resulted in a rapid increase in surface pressure of the glycolipid/lipid films, thus indirectly confirming their interactions with GalCer, GlcCer, LacCer, and ceramide. In summary, we show that HIV and SIV envelope glycoproteins bind to GalCer, GlcCer, LacCer, and ceramide in a dose-dependent, saturable, and specific manner. These interactions may function as receptors of attachment in order to facilitate infection of CD4 low or negative cells or promote interactions with other receptors leading to the activation of signaling pathways or pathogenesis.

Synthesis and biological activities of amino acids functionalized α-GalCer analogues.

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6' position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain.

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes.

Lewis Acid Induced Anomerization of Se-Glycosides. Application to Synthesis of α-Se-GalCer.

The TiCl4 induced anomerization of selenium glycosides of galacturonic acid derivatives is reported. The reaction was successful for galacturonic acid when various alkyl, alkenyl, alkynyl, saccharide, steroid, and lipid groups were attached to the anomeric Se atom. An increased amount of TiCl4 and/or higher temperature were needed to ensure completion of the reaction in some cases. Yields were higher for reactions carried out at higher dilution. The reaction was applied to the synthesis of Se-based mimics of the potent immunostimulant α-GalCer (KRN7000).

Synthesis of 6"-triazole-substituted α-GalCer analogues as potent iNKT cell stimulating ligands.

We report the synthesis of a small series of 6"-triazol-1-yl-substituted α-GalCer analogues by late-stage conversion of the 6"-OH to an azide group, copper-catalyzed azide-alkyne cycloaddition and final deprotection. When evaluated for their capacity to induce IL-2 secretion in vitro, all compounds proved equally potent or superior to α-GalCer. The S.A.R suggests that the improved antigenic activity is mainly triggered by the triazole functionalization in se. While the introduction of selected substitutuents at C-4 of this heterocyclic ring is tolerated, this generally fails to further improve antigenicity.

Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains.

We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ~1 Hz with a sensitivity in terms of surface coverage of ~1 ng/cm(2). Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.

Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides.

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of ß-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 µM range with no toxicity to the cells at concentrations up to 200 µM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

Catalytic Z-selective olefin cross-metathesis for natural product synthesis.

Alkenes are found in many biologically active molecules, and there are a large number of chemical transformations in which alkenes act as the reactants or products (or both) of the reaction. Many alkenes exist as either the E or the higher-energy Z stereoisomer. Catalytic procedures for the stereoselective formation of alkenes are valuable, yet methods enabling the synthesis of 1,2-disubstituted Z alkenes are scarce. Here we report catalytic Z-selective cross-metathesis reactions of terminal enol ethers, which have not been reported previously, and of allylic amides, used until now only in E-selective processes. The corresponding disubstituted alkenes are formed in up to >98% Z selectivity and 97% yield. These transformations, promoted by catalysts that contain the highly abundant and inexpensive metal molybdenum, are amenable to gram-scale operations. Use of reduced pressure is introduced as a simple and effective strategy for achieving high stereoselectivity. The utility of this method is demonstrated by its use in syntheses of an anti-oxidant plasmalogen phospholipid, found in electrically active tissues and implicated in Alzheimer's disease, and the potent immunostimulant KRN7000.

A rapid fluorescence-based assay for classification of iNKT cell activating glycolipids.

Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells.

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines.

Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)] of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar derivatives with a cyclic sulfamidate (9) as the key step. Bioassay showed RCAI-56 (3, carbagalactose analog of KRN7000), 59 (5, 1-deoxy-neo-inositol analog), and 92 (7, 1-O-methylated 5) to be remarkably potent stimulants of mouse lymphocytes to produce Th1-biased cytokines, such as interferon-gamma, in vivo. RCAI-60 (4, carbafucose analog) and RCAI-101 (6, 6-O-methylated 3) showed strong bioactivity, on the other hands, RCAI-37 (1, myo-inositol analog) and 102 (2, neo-inositol analog) induced little cytokine production.

Dendritic catanionic assemblies: in vitro anti-HIV activity of phosphorus-containing dendrimers bearing galbeta1cer analogues.

Two series of water-soluble dendritic catanionic assemblies, acting as multisite analogues of galactosylceramide (Galbeta1cer), have been prepared with the goal of blocking HIV infection prior to the entry of the virus into human cells. Trifunctional and hexafunctional cinnamic acid-terminated dendrimers have been synthesized from phosphorus-containing dendrimers bearing aldehyde end groups. A classical acid-base reaction performed in water between acid-terminated dendrimers and stoichiometric amounts of N-hexadecylamino-1-deoxylactitol provided the expected catanionic assemblies. Antiviral assays on these supramolecular entities confirmed the crucial roles both of multivalency effects and of lipophilicity on the biological activity of Galbeta1cer analogues. Moreover, correlation between in vitro tests and molecular modeling highlights the specific influence of the assembly shape on the anti-HIV efficiency, with the tri- and hexafunctional cored dendrimers, both decorated with 12 sugar moieties, exhibiting IC50 values of 1.1 and 0.12 microM, respectively.

Phosphorus-containing dendrimers bearing galactosylceramide analogs: self-assembly properties.

We report the synthesis and the supramolecular auto-assembly of catanionic phosphorus-containing dendrimers mimicking multisite analogs of galactosylceramide.

Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase.

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.