Sung Kee Chung

Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate.

Ca2+-activated Cl− channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092–14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4–2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4–2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3,4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 μm). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to “switch-off” the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 μm, respectively). The non-physiological trisphosphates,d/l-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 μm), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (±)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates☆

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

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.

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.

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.

Photolytic Cleavage and Condensation Reactions of Cyclohexa-2,4-dienones with Diamines

Cyclohexa-2,4-diene-1-one sulfone derivate undergoes ring cleavage to afford bis-amides containing a diene moiety on irradiation with visible light in the presence of various diamines.

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.

A Facile One-Step Synthesis of Ethyl 2-(L,L-Dialkyl and Arylmethyl) Malonates

Abstract Diethyl ethoxymethylenemalonate (EMME) is unexpectedly converted into diethyl 2-(1,1 -dialkyl and diarylmethyl) malonate derivatives by a classical Grignard reaction. Addition of organocuprates to EMME gave diethyl 2-(l-ethoxyalkyl) malonate derivatives. The yields range from 42-63%.

Photochemically induced symmetrical coupling reactions between cyclohexa-2,4-dienones and α,ω-diamines. A new approach to the selective labelling of peptides and proteins

Abstract 2,4,6-Trimethylcyclohexa-2,4-dien-1-one derivatives undergo ring cleavage to furnish symmetrical amides on irradiation with visible light in the presence of various α,ω-diamines. Five different symmetrical amides containing a substituted diene moiety were synthesized in 65–85% yield.

Synthesis of thiophenyl substituted cyclohexa-2,4-dien-1-one and its photocleavage coupling reaction with amines

Thiophenyl substituted cyclohexa-2,4-dien-1-ones were synthesized and photolyzed in the presence of various amines to afford the amides containing diene moeties via the ketene intermediate under visible light irradiation at 38 degrees C.