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.

Elimination of a hydroxyl group in FTY720 dramatically improves the phosphorylation rate.

The new immunosuppressant FTY720 (fingolimod), an analog of the endogenous lipid sphingosine, induces transient lymphopenia through the sequestration of lymphocytes in secondary lymphoid organs. Phosphorylation of FTY720 by sphingosine kinase 2 (SphK2) yields the active metabolite FTY720-phosphate (FTY-P), which induces lymphopenia through agonism of the sphingosine 1-phosphate receptor S1P(1) on endothelial cells and lymphocytes. Dephosphorylation of circulating FTY-P creates an equilibrium between FTY720 and its phosphate, and results with human patients indicate that phosphorylation of FTY720 could be rate limiting for efficacy. We report that the FTY720 derivative 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] is phosphorylated much more rapidly than FTY720 in cultured human cells and whole blood. The K(cat) for AAL(R) with recombinant SphK2 is 8-fold higher than for FTY720, whereas the K(m) for the two substrates is very similar, indicating that the increased rate of phosphorylation results from faster turnover by SphK2 rather than a higher binding affinity. Consequently, treating cells with AAL(R), but not FTY720, triggers an apoptotic pathway that is dependent on excessive intracellular accumulation of long-chain base phosphates. In agreement with the in vitro results, phosphorylation of AAL(R) is more complete than that of FTY720 in vivo (mice), and AAL(R) is a more potent inducer of lymphopenia. These differences may be magnified in humans, because phosphorylation of FTY720 is much less efficient in humans compared with rodents. Our results suggest that AAL(R) is a better tool than FTY720 for in vivo studies with S1P analogs and would probably be a more effective immunosuppressant than FTY720.

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.

Chemoenzymatic syntheses of carbasugar analogues of nucleoside diphosphate sugars: UDP-carba-Gal, UDP-carba-GlcNAc, UDP-carba-Glc, and GDP-carba-Man.

Chemoenzymatic syntheses of several NDP-carba-sugars have been successfully carried out, and these essential cofactor analogues are expected to be selective inhibitors of glycosyltransferase enzymes.

Spatial expression patterns and biochemical properties distinguish a second myo-inositol monophosphatase IMPA2 from IMPA1.

Lithium is used in the clinical treatment of bipolar disorder, a disease where patients suffer mood swings between mania and depression. Although the mode of action of lithium remains elusive, a putative primary target is thought to be inositol monophosphatase (IMPase) activity. Two IMPase genes have been identified in mammals, the well characterized myo-inositol monophosphatase 1 (IMPA1) and myo-inositol monophosphatase 2 (IMPA2). Several lines of genetic evidence have implicated IMPA2 in the pathogenesis of not only bipolar disorder but also schizophrenia and febrile seizures. However, little is known about the protein, although it is predicted to have lithium-inhibitable IMPase activity based on its homology to IMPA1. Here we present the first biochemical study comparing the enzyme activity of IMPA2 to that of IMPA1. We demonstrate that in vivo, IMPA2 forms homodimers but no heterodimers with IMPA1. Recombinant IMPA2 exhibits IMPase activity, although maximal activity requires higher concentrations of magnesium and a higher pH. IMPA2 shows significantly lower activity toward myo-inositol monophosphate than IMPA1. We therefore screened for additional substrates that could be more efficiently dephosphorylated by IMPA2, but failed to find any. Importantly, when using myo-inositol monophosphate as a substrate, the IMPase activity of IMPA2 was inhibited at high lithium and restricted magnesium concentrations. This kinetics distinguishes it from IMPA1. We also observed a characteristic pattern of differential expression between IMPA1 and IMPA2 in a selection of tissues including the brain, small intestine, and kidney. These data suggest that IMPA2 has a separate function in vivo from that of IMPA1.