Synthesis and evaluation of sphinganine analogues of KRN7000 and OCH

[structures: see text] The phytosphingosine-containing alpha-galactosylceramides (alpha-GalCers), KRN7000 and OCH, have been shown to activate NKT cells via interaction with CD1d, a member of the CD1 family of antigen presenting proteins. Evidence from KRN7000 stimulation of NKT cells suggests that alpha-GalCers may have applications in the treatment or prevention of a range of viral, bacterial, and autoimmune conditions. Moreover, OCH, a truncated analogue of KRN7000, appears to induce a T(H)2 bias, which could have implications for the treatment of autoimmune and inflammatory conditions. We have prepared the direct sphinganine-containing analogues of KRN7000 and OCH, 1 and 2, and found them to be comparable in activity to the parent compounds in inducing the release of IL-2, IL-4, and IFNgamma. In addition, compound 2 leads to a cytokine bias similar to that seen with OCH. This is significant because sphinganines are more easily accessed than phytosphingosines, which should facilitate SAR studies.     

Molecular dynamics simulation study on the interaction of KRN 7000 and three analogues with human CD1d

Many analogues of KRN 7000, a synthetic glycolipid (α-galactosylceramide) exhibiting immuno-stimulatory activity and antitumor properties, were previously synthesized and tested in order to understand the reasons for the resulting biological activity and Th1/Th2 cytokine profile. Principles have been established for the interaction of such glycolipids with the human CD1d molecule but the exact mechanism by which different ligands with the same polar head elicit distinct biological responses remains unclear. Based on these experiments and on the available crystal structures, protein-ligand interactions are explored using molecular dynamics simulations. Hydrogen bond interactions are examined with regard to the polar group orientation. The influence of modulations on the dynamic behavior of the CD1d-glycolipid complex is addressed. Overall, our data support the mechanism by which the shortening of the α-GalCer sphingosine chain causes a significant twist of the CD1d α1 helix structure from residue Phe84 that affects the position of CD1d residues involved in the TCR recognition.     

Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production

The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates T(H)1 and T(H)2 chemokine release. In this study, a variety of glycolipid ligands were attached to a microarray surface and their binding with dimeric CD1d was investigated. An alpha-galactosyl ceramide (alpha-GalCer) bearing a carbamate group at the 6'-OH position was tethered to the surface, and the dissociation constant on surface with CD1d was determined to reflect the multivalent interaction. Competition assays were then used to determine the dissociation constants (Ki) of new and intact glycolipids in solution. The 4-fluorophenyloctanoyl-modified alpha-GalCer (18) was found to bind most strongly with CD1d (Ki 0.21 microM), 2 orders of magnitude stronger than alpha-GalCer and more than three times more selective than alpha-GalCer for IFN-gamma release from NKT cells. Various alpha-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-gamma production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the T(H)1 pathway.     

The stimulating adventure of KRN 7000

Associated with the CD1d protein, KRN 7000, a potent synthetic α-galactosylceramide, is known to activate the invariant NKT immune cells. This stimulation then leads to the production of different cytokines modulating a T(H)1/T(H)2 immune response balance involved in protection against several pathologies such as autoimmune diseases and cancers. Various efforts have been made toward the synthesis of simple and more functionalized analogues in order to selectively induce T(H)1 or T(H)2-type cytokine production. Since the discovery of KRN 7000, structure-activity relationships, crystallographic and modelling studies have pointed to the potential of several GalCer analogues in term of selective bioactivity, and have highlighted interesting elements in order to better understand the recognition and activation mechanisms of immune iNKT cells. By presenting an up-to-date library of analogues, collecting recent breakthroughs done in crystallography and molecular modelling, and relating them to the available biological results, we hope that this review will highlight and help the scientific community in their KRN research.     

Divergent synthetic approach to 6'-modified α-GalCer analogues

A synthetic approach is presented for the synthesis of galacturonic acid and D-fucosyl modified KRN7000. The approach allows for late-stage functionalisation of both the sugar 6'-OH and the sphingosine amino groups, which enables convenient synthesis of promising 6'-modified KRN7000 analogues.     

Synthesis of tritium labeled KRN7000

The synthesis of a multiple tritiated analog of KRN7000 (α-galactosyl ceramide) is described, enabling further studies to quantitate the affinity of KRN7000 for its receptor and to study its pharmacokinetic properties.     

RCAI-56, a carbocyclic analogue of KRN7000: its synthesis and potent activity for natural killer (NK) T cells to preferentially produce interferon-γ

RCAI-56 (3), a carbocyclic analogue of KRN7000 (1) was synthesized through an efficient coupling of a carba-α-d-galactose derivative 11 with cyclic sulfamidate derivative 13 of phytosphingosine to give 15. Carbasugar derivative 11 was prepared by starting from methyl α-d-galactopyranoside (4), employing Pd(II)-catalyzed Ferrier rearrangement as the key step. RCAI-56 (3) is a potent stimulant of NKT cells in vivo to induce the production of Th1 biased cytokines such as interferon-γ in mice. According to the docking model of CD1d-3 complex, its stabilization energy is approximately at the same level as that of the CD1d-1 complex.     

RCAI-61, the 6′-O-methylated analog of KRN7000: its synthesis and potent bioactivity for mouse lymphocytes to produce interferon-γ in vivo

RCAI-61, the 6′-O-methylated analog of KRN7000, and six other analogs with modified 6′-position of the galactose moiety of KRN7000 were synthesized to examine their bioactivity for mouse lymphocytes. Methyl α-d-galactopyranoside was the starting material for RCAI-58, 61, 64, 83, 85, and 86, while RCAI-87 was prepared from methyl β-l-arabinopyranoside. Bioassay showed RCAI-61 to be a much more potent stimulant of mouse lymphocytes than KRN7000 and RCAI-56 to induce the production of a large amount of IFN-γ in vivo.     

Synthesis and biological activity of hydroxylated analogs of RCAI-80

RCAI-80 is one of the ester analogs of KRN7000 (α-galactosylceramide). This compound released mainly T helper 2 (Th2) cytokines, such as IL-4 rather than T helper 1 (Th1) cytokines, such as IFNγ from the invariant natural killer T (iNKT) cells. In addition, it has been known that some of the hydroxylated derivatives of KRN7000 make the cytokine secretion bias to Th2 by decreasing the IFNγ production to almost zero. This time, the three compounds having these two characteristic properties, namely an ester group and also some extra hydroxy groups existing on the ester side chain and/or on the 2-acyloxy-3,4-dihydroxyoctadecyl main chain of RCAI-80, were synthesized to examine the biological activities. As a result, it was found that these compounds made the cytokine secretion skew to Th2. Therefore, their effectiveness for experimental autoimmune encephalomyelitis (EAE) was examined. It was recognized that one of them showed moderate suppression of EAE symptom.     

The first synthesis of a thioglycoside analogue of the immunostimulant KRN7000

The first total synthesis of a thioglycoside analogue of KRN7000, a potential immunostimulant, is described. Two key intermediates are alpha-galactosyl thiol 4 and phytosphingosine derivative 5, which were both prepared from D-galactose.     

Synthesis of alpha-galactosyl ceramide (KRN7000) and analogues thereof via a common precursor and their preliminary biological assessment

A new practical synthesis of alpha-GalCer and of its analogues is presented, opening the chance to easily modify the sphingosine chain. The common precursor is a disaccharide, obtained by coupling tetra-O-benzyl-D-galactose with allyl 2,3-O-isopropylidene-D-lyxofuranoside. Introduction of alkyl chains via Wittig reaction (for alpha-GalCer and OCH) or via Williamson reaction (for oxa analogues) followed by standard synthetic steps allows one to efficiently obtain such compounds. The analogues are able to activate iNKT cells when presented by CD1d expressing cells.     

Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure–activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.     

Concise syntheses of immunostimulating glycolipids, α-galactosyl ceramides

α-Galactosylceramides (α-GalCers) are well known as immunostimulating agents having therapeutic potential. To facilitate the synthesis of α-GalCers and their derivatives, a novel and convergent strategy was designed, which is expected to be versatile for the preparation of a variety of analogues in a diversity-oriented fashion. As an initial demonstration of our strategy, KRN7000 and OCH were synthesized in eight steps from a common intermediate, which is easily obtainable in a multi-gram scale.     

Expedient synthesis of the alpha-C-glycoside analogue of the immunostimulant galactosylceramide (KRN7000)

[Structure: see text] Key reactions in a concise synthesis of an alpha-C-galactosylceramide analogue of KRN7000 include a diastereoselective alkenylalane addition to an N-tert-butanesulfinyl imine and the use of an epoxidation/carbamate ring opening sequence to install the aminodiol stereotriad.     

Total synthesis of α-1C-galactosylceramide, an immunostimulatory C-glycosphingolipid, and confirmation of the stereochemistry in the first-generation synthesis

A nonisosteric α-C-glycoside analogue of KRN7000 (α-1C-GalCer, 1) was reported to induce a selective type of cytokine release in human invariant natural killer cells in vitro. We report here a very concise synthetic route to 1 and its analogue 1'. The key steps include olefin cross-metathesis, Sharpless asymmetric epoxidation, and epoxide opening by NaN(3)/NH(4)Cl. Inversion of configuration at the amide-bearing carbon in the phytosphingosine backbone constructed by epoxide opening in our previous synthesis of 1 was verified, indicating that remote group participation is not involved during the epoxide-opening reaction.     

Synthesis and anti-tumor activity of β-C-glycoside analogs of the immunostimulant KRN7000

A ring-closing metathesis approach was employed for the synthesis of a β-C-glycoside analog of the immunostimulant KRN7000. The protected C-glycosyl amino acid derivative 18 was converted to amino-olefin 20, and osmylation served to install the diol unit as a mixture of separable syn and anti isomers. Deprotection to the hydroxy-amine 21 was followed by N-acylation and debenzylation to deliver the target compound 5.     

Structure-based discovery of glycolipids for CD1d-mediated NKT cell activation: tuning the adjuvant versus immunosuppression activity

Introduction of an aromatic group into the fatty acyl chain of alpha-GalCer modulates the activity and selectivity of IFN-gamma/IL-4 secretion through CD1d-mediated activation of NKT cells. Compound 14-16 are more potent than alpha-Galcer and biased for IFN-gamma than for IL-4. These new glycolipids may find use as adjuvants or as antimetastatic agents.     

Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.     

Binding of alkaloids into the S1 specificity pocket of α-chymotrypsin: evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K(a) ≈ 10(5) M(-1)) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K(a) ≈ 10(4) M(-1)). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthermore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases.     

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.