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.

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.

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.

Recent advances in cell-penetrating, non-peptide molecular carriers.

The intracellular delivery of proteins and other bioactive molecules by employing membrane-permeable carrier peptide vectors, e.g. HIV-1 Tat, Antp-HD, and related arginine-rich peptides are well known for a number of years. Because of some real and potential problems associated with these peptide carriers, such as instability due to various endogenous peptidases, uncertain in vivo delivery efficiency, potential neurotoxicity and immunogenicity, an urgent need exists for the development of efficient, non-peptide molecular carriers. This review briefly summarizes the structural characteristics and the delivery properties of the newly developed non-peptide carriers, in particular the ones developed in the author's laboratory, together with their potential as delivery vectors for poorly bioavailable drugs including small molecules, proteins, and nucleotides.

Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase.

Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases.

Intracellular and transdermal protein delivery mediated by non-covalent interactions with a synthetic guanidine-rich molecular carrier.

The impermeability of the cell plasma membrane is one of the major barriers for protein transduction into mammalian cells, and it also limits the use of proteins as therapeutic agents. Protein transduction has usually been achieved based on certain invasive processes or cell penetrating peptides (CPP). Herein we report our study in which a synthetic guanidine-rich molecular carrier is used as a delivery vector for intracellular and transdermal delivery of proteins. First a sorbitol-based molecular carrier having 8 guanidine units (Sor-G8) was synthesized, and then was simply mixed with a cargo protein of varying sizes to form the non-covalent complex of carrier-cargo proteins. These ionic complexes were shown to have efficient cellular uptake properties. The optimum conditions including the molar ratio between cargo protein and carrier, and the treatment time have been defined. Several protein cargoes were successfully examined with differing sizes and molecular weights: green fluorescent protein (MW 27kDa), albumin (66kDa), concanavalin A (102kDa), and immunoglobulin G (150kDa). These non-covalent complexes were also found to have excellent transdermal penetration ability into the mouse skin. The skin penetration depth was studied histologically by light microscopy as well as two-photon microscopy thus generating a depth profile. These complexes were largely found in the epidermis and dermis layers, i.e. down to ca. 100µm depth of the mouse skin. Our synthetic Sor-G8 carrier was found to be substantially more efficient that Arg8 in both the intracellular transduction and the transdermal delivery of proteins. The mechanism of the cellular uptake of the complex was briefly studied, and the results suggested macropinocytosis.

A guanidine-appended scyllo-inositol derivative AAD-66 enhances brain delivery and ameliorates Alzheimer's phenotypes.

Alzheimer's disease (AD) is a degenerative brain disease that destroys memory and other important mental functions but lacks efficient therapeutic agents. Blocking toxic amyloid ß (Aß) could be beneficial for AD and represents a promising therapeutic strategy for AD treatment. scyllo-Inositol (SI) is a potential therapeutic for AD by directly interacting with the Aß peptide to inhibit Aß42 fiber formation. Clinical studies of SI showed promising benefits on mild to moderate AD, however, with limitations on dosage regime. A new strategy to enhance the brain delivery of SI is needed to achieve the efficacy with minimum adverse effects. Herein, we report that a novel guanidine-appended SI derivative AAD-66 resulted in more effective reductions of brain Aß and plaque deposits, gliosis, and behavioral memory deficits in the disease-established 5xFAD mice. Overall, our present study reveals the potential of AAD-66 as a promising therapeutic agent for AD.

On the contribution of stereochemistry to human ITPK1 specificity: Ins(1,4,5,6)P4 is not a physiologic substrate.

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

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.

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds.

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.

Vaccinia-related kinase 2 mediates accumulation of polyglutamine aggregates via negative regulation of the chaperonin TRiC.

Misfolding of proteins containing abnormal expansions of polyglutamine (polyQ) repeats is associated with cytotoxicity in several neurodegenerative disorders, including Huntington's disease. Recently, the eukaryotic chaperonin TRiC hetero-oligomeric complex has been shown to play an important role in protecting cells against the accumulation of misfolded polyQ protein aggregates. It is essential to elucidate how TRiC function is regulated to better understand the pathological mechanism of polyQ aggregation. Here, we propose that vaccinia-related kinase 2 (VRK2) is a critical enzyme that negatively regulates TRiC. In mammalian cells, overexpression of wild-type VRK2 decreased endogenous TRiC protein levels by promoting TRiC ubiquitination, but a VRK2 kinase-dead mutant did not. Interestingly, VRK2-mediated downregulation of TRiC increased aggregate formation of a polyQ-expanded huntingtin fragment. This effect was ameliorated by rescue of TRiC protein levels. Notably, small interference RNA-mediated knockdown of VRK2 enhanced TRiC protein stability and decreased polyQ aggregation. The VRK2-mediated reduction of TRiC protein levels was subsequent to the recruitment of COP1 E3 ligase. Among the members of the COP1 E3 ligase complex, VRK2 interacted with RBX1 and increased E3 ligase activity on TRiC in vitro. Taken together, these results demonstrate that VRK2 is crucial to regulate the ubiquitination-proteosomal degradation of TRiC, which controls folding of polyglutamine proteins involved in Huntington's disease.

Preparation of a 3'-azido-3'-deoxythymidine (AZT) derivative, which is blood-brain barrier permeable.

The anti-AIDS drug AZT was covalently attached to the recently reported sorbitol-G8 transporter, and the conjugate was found to target mitochondria in HeLa cells and readily cross the blood-brain barrier (BBB) to gain access into the mouse brain, suggesting the potential of these molecular transporters as practical central nervous system (CNS) delivery vectors.

Novel lipidated sorbitol-based molecular transporters for non-viral gene delivery.

In this study, we investigated the possible use of novel lipidated sorbitol-based transporters as functional devices for the improvement of non-viral gene delivery. These transporters are composed of a sorbitol scaffold bearing 8 guanidine moieties that mimic the arginine residues of well-known cell-penetrating peptides. In addition, the transporters carry different lipid groups to aid DNA condensation and facilitate lipid vesicle-binding. We found that the transporters described in this study have the potential to function as plasmid DNA/siRNA-condensers and surface ligands for the enhancement of cellular uptake of lipid vesicles. Shorter lipid chains were found to be better for condensation, whereas longer chains were superior surface ligands. The differential activity of different cores might be explained by facilitated decondensation of cores prepared with transporters comprised of shorter lipid chains. However, we suggest that there is an optimum value of decondensation to achieve higher transfection activities. The proper use of the transporters presented in this study enabled us to prepare a highly efficient non-viral gene delivery system based on a core-shell structure, in which a condensed DNA core is encapsulated by a lipid envelope. A multifunctional envelope-type nano-device prepared with an optimal surface ligand favorably competes with commonly used transfection systems.

Synthesis and cellular uptake properties of guanidine-containing molecular transporters built on the sucrose scaffold.

Novel sucrose-based G7 molecular transporters show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes.