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.

Syntheses and biological activities of KRN7000 analogues having aromatic residues in the acyl and backbone chains with varying stereochemistry.

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release a broad range of bioactive cytokines. In an effort to understand the structure-activity relationships, we have carried out syntheses of 26 new KRN7000 analogues incorporating aromatic residues in either or both side chains. Structural variations of the phytosphingosine moiety also include varying stereochemistry at C3 and C4, and 4-deoxy and 3,4-dideoxy versions. Their biological activities are described.

Evaluation of synthetic sphingolipid analogs as ligands for peroxisome proliferator-activated receptors.

In this Letter, we assessed newly synthesized sphingolipid analogs as ligands for peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta or PPARgamma, using a dual-luciferase reporter system. We tested 640 sphingolipid analogs for ligand activity. As a result, seven types: A9, B9, C9, C50, F66, G66 and H66, were found to show agonistic activities for PPARs.

Alternated Branching Ratios by Anomaly in Collision-Induced Dissociation of Proton-Bound Hoogsteen Base Pairs of 1-Methylcytosine with 1-Methylguanine and 9-Methylguanine.

A comparative study on the proton-bound complexes of 1-methylcytosine (1-mC) with 1-methylguanine (1-mG) and 9-methylguanine (9-mG), [1-mC:1-mG:H]+ and [1-mC:9-mG:H]+, respectively, was carried out using energy-resolved collision-induced dissociation (ER-CID) experiments in combination with quantum chemical calculations. In ER-CID experiments, the measured survival yields indicated an essentially identical stability for the two proton-bound complexes. In comparison with the lowest-energy structures and base-pairing energetics predicted at the B3LYP/6-311+G(2d,2p) theory level, both complexes produced in this study were suggested to be proton-bound Hoogsteen base pairs. Curiously, despite the similarity in structures, binding energetics, and potential energy surfaces predicted by the B3LYP theory, the fragment branching ratios exhibited an intriguing alternation between the two proton-bound Hoogsteen base pairs. The CID of [1-mC:1-mG:H]+ produced protonated cytosines, [1-mC:H]+, more abundantly than [1-mG:H]+, whereas that of [1-mC:9-mG:H]+ gave rise to a more pronounced production of protonated guanines, [9-mG:H]+. However, using the proton affinities of moieties predicted by the high-accuracy methods, including CBS-QB3 and the Guassian-4 theory, the anomaly known for [Cytosine:Guanine:H]+ (J. Am. Soc. Mass Spectrom. 29, 2368-2379 (2018)) successfully accounted for the alternated branching ratios. Thereby, the anomaly, more specifically, the production of proton-transferred fragments of O-protonated cytosines in the CID of proton-bound Hoogsteen base pairs, is indeed real, which is disclosed as the alternated branching ratios in the CID spectra of [1-mC:1-mG:H]+ and [1-mC:9-mG:H]+ in this study. Graphical Abstract .

Synthesis of all stereoisomers of KRN7000, the CD1d-binding NKT cell ligand.

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure-activity relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers, and their biological activities have been examined.

Divergent syntheses of all stereoisomers of phytosphingosine and their use in the construction of a ceramide library.

Sphingolipids such as ceramide and sphingosine-1-phosphate have recently attracted intense research interests because of their functional roles as signaling molecules in many important physiological processes, such as growth arrest, apoptosis, and inflammatory responses, and cell proliferation, vascular maturation and trafficking of lymphocytes. The well-defined modular structures of ceramides and related glycosylceramides are ideally amenable to library formation for medicinal chemistry investigation. We have developed divergent synthetic routes to all eight phytosphingosine stereoisomers and then proceeded to prepare phytosphingosine-based ceramide library composed of more than 500 compounds.

Proton Transfer Accounting for Anomalous Collision-Induced Dissociation of Proton-Bound Hoogsteen Base Pair of Cytosine and Guanine.

To understand the anomalous collision-induced dissociation (CID) behavior of the proton-bound Hoogsteen base pair of cytosine (C) and guanine (G), C:H+∙∙∙G, we investigated CID of a homologue series of proton-bound heterodimers of C, 1-methylcytosine, and 5-methylcytosine with G as a common base partner. The CID experiments were performed in an energy-resolved way (ER-CID) under both multiple and near-single collision conditions. The relative stabilities of the protonated complexes examined by ER-CID suggested that the proton-bound complexes produced by electrospray ionization in this study are proton-bound Hoogsteen base pairs. On the other hand, in contrast to the other base pairs, CID of C:H+∙∙∙G exhibited more abundant productions of C:H+, the fragment protonated on the moiety with a smaller proton affinity, than that of G:H+. This appeared to contradict general prediction based on the kinetic method. However, further theoretical exploration of potential energy surfaces found that there can be facile proton transfers in the proton-bound Hoogsteen base pairs during the CID process, which makes the process accessible to an additional product state of O-protonated C for C:H+ fragments. The presence of an additional dissociation channel, which in other words corresponds to twofold degeneracy in the transition state leading to C:H+ fragments, effectively doubles the apparent reaction rate for production of C:H+. In this way, the process gives rise to the anomaly, the observed pronounced formation of C:H+ in the CID of the proton-bound Hoogsteen base pair, C:H+∙∙∙G. Graphical Abstract ᅟ.

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

Syntheses of sphingosine-1-phosphate analogues and their interaction with EDG/S1P receptors.

Sphingosine-1-phosphate (S1P) is an important regulator of a wide variety of biological processes acting as an endogenous ligand to EDG/S1P receptors. In an effort to establish structure-activity relationship between EDG/S1P and ligands, we report herein homology modeling study of EDG-1/S1P(1), syntheses of S1P analogues, and cell based binding affinity study for EDG/S1P receptors.