Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.FTY720 is derived from ISP-1 (myriocin), a fungal metabolite that is an eternal youth nostrum in traditional Chinese herbal medicine (1). The compound (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol) is a novel, high potency immune modulating agent that is remarkably effective in a variety of autoimmune and transplant models including islet transplantation (2) and has recently proven to be effective in renal transplantation in man (3). Unlike the currently used immunosuppressive agents (e.g. the calcineurin inhibitors cyclosporin and tacrolimus), FTY720 does not inhibit T cell activation and proliferation and in rodent models does not impair immunity to systemic viral infection (4). If confirmed in man, the latter property provides a striking advantage over current immunosuppressive therapies. FTY720 apparently sequesters lymphocytes from circulation to secondary lymph tissue compartments (5) with concomitant reduction of specific effector T cells recirculating from the lymph nodes to inflamed peripheral tissues (4) and graft sites (6). FTY720 does not act via the lymphocytehoming chemokine receptor CCR-7 because FTY720 is active both in CCR-7-deficient mice and plt (paucity of lymph node T cells) mice, which lack CCR-7 ligands (CCL-19 and CCL-21) (7).FTY720-induced lymphocyte homing is sensitive to suppression by pertussis toxin (6 -8), which suggests that the molecular target of the drug is a G protein-coupled receptor (GPCR) 1 interacting with heterotrimeric G proteins of the ␣ i/o type. The affected GPCR(s) is on the lymphocyte since fluorescently labeled lymphocytes treated with pertussis toxin ex vivo and transferred to mice are not depleted by FTY720 in vivo (8). The structural similarity of FTY720 and sphingosine has prompted speculation that the drug might act via the sphingosine 1-phosphate (S1P) receptor S1P 4 (formerly 2 that is known to be expressed by lymphocytes (9). S1P is a pleiotropic lysophospholipid mediator; the prominent cellular responses to applied S...

The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of sphingosine 1-phosphate (S1P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower K m of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.FTY720 is an immunomodulatory drug, which is highly efficacious in models of transplantation and of autoimmune diseases (1). It was recently found to be effective in kidney transplantation in humans (2). FTY720 elicits a lymphopenia resulting from the reversible redistribution of lymphocytes from the circulation to secondary lymphoid organs, without leading to general immunosuppression (3, 4). Conversion of FTY720 to its monophosphate appears to be essential for the effects of the drug on lymphocyte homing, since FTY720 phosphate acts as an agonist at four of the five G-protein-coupled receptors for sphingosine-1-phosphate (S1P) 1 (5, 6); it is assumed that at least one S1P receptor is critical to the lymphopenic response induced by FTY720 treatment (2). More recently, FTY720 was found to stimulate multidrug transporterdependent T-cell chemotaxis to lymph nodes (7); in this instance, FTY720 phosphate as the active metabolite is hypothesized to be responsible for stimulation of efflux activity of the lipid transporter Abcb1. FTY720 has been reported to be phosphorylated ex vivo by rodent lymphoid tissues (5) and whole blood of several species (6), and is rapidly phosphorylated in vivo (5, 6). After oral application of FTY720 to rats, the blood levels of the monophosphate exceeded those of the parent compound 2-4 fold (5). FTY720 was shown to be a substrate for recombinant murine sphingosine kinase 1a (muSPHK1a) (5). Studies with chiral analogs of FTY720 (namely the R-an...

FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the sphingosine-1-phosphate receptor S1P 1 is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of sphingosine kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 phosphate formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 phosphate, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 phosphate levels in vivo. IntroductionNaive T cells regularly circulate between the bloodstream and lymphatic tissue in search for foreign antigen, as well as for tumor and autoantigen. Their activation in secondary lymphoid organs followed by regulated egress back into the circulation to reach sites of inflammation is a prerequisite for any adaptive immune response in the T-cell compartment. Recently, one of the G protein-coupled receptors for sphingosine-1-phosphate (S1P), namely the S1P 1 receptor, was shown to be crucial for the tempo-spatial trafficking of T cells into and out of the secondary peripheral lymphoid organs. 1 The importance of S1P 1 in lymphocyte trafficking became clear through studies with FTY720, an analog of sphingosine. FTY720, after phosphorylation in vivo to FTY720 phosphate (FTY720-P), induces a reversible sequestration of lymphocytes into lymph nodes and Peyer patches. 2,3 FTY720-P thereby acts as a functional antagonist of the S1P 1 receptor, thus inducing aberrant internalization and consequently rendering T cells unresponsive to the obligatory egress signal provided by S1P. 1,[4][5][6] FTY720 has emerged as a potent immunomodulatory agent with usefulness in the control of organ transplant rejection and for treatment of autoimmune diseases. In animals, FTY720 is efficacious in prolonging graft survival, as well as in models of multiple sclerosis, acute lung injury, autoimmune diabetes, atherosclerosis, and renal ischemia-reperfusion injury. 7 Promising results have been obtained from human trials on FTY720 for indications in renal transplantation and multiple sclerosis. 7,8 Since FTY720 prodrug activation is essential for its action on T (and B) cells, understanding how the drug gets phosphorylated in vivo is of high interest, in particular for the design of novel analogs with altered pharmacologic properties.FTY720 is known to be phosphorylated in vitro by the 2 mammalian sphingosine kinases SPHK1 and 2, with SPHK2 being considerably more efficient. [9][10][11] As shown by a recent study in SPHK1-deficient mice, 12 this enzyme appears to be dispensable for the action of FTY720 in vivo, as Sphk1-null mice are still rendered lymphopenic by the drug.In this study, we describe th...

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to Fc⑀RI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.Receptor stimulation of a variety of cell types induces the early activation of sphingosine kinase (SphK), 5 a unique lipid kinase that generates the potent and versatile lipid mediator sphingosine-1-phosphate (S1P) (1, 2). S1P was demonstrated to function intracellularly, regulating cell survival, cell proliferation, and calcium fluxes (1, 3). However, many of the effects of S1P can be attributed to its role as a ligand for a family of five G-protein-coupled receptors, named the endothelial differentiation gene or S1P receptors (S1P 1-5 ) (4, 5). This receptor family, and predominantly the S1P 1 subtype, is known to regulate diverse biological functions, including cytoskeletal changes, chemotaxis, vascular development, regulation of endothelial cell function, and lymphocyte recirculation (1, 4). Two distinct mammalian SphKs (SphK1 and SphK2) have been identified, and both kinases efficiently convert sphingosine to S1P (6, 7). SphK1 is activated in response to multiple stimuli, whereas SphK2 activation was only recently demonstrated in response to epidermal growth factor (2,8,9).In mast cells, the high affinity receptor for IgE (Fc⑀RI)-mediated activation of SphK1 has been associated with calcium mobilization and degranulation (10 -12). However, the intracellular role of S1P in modulating mast cell calcium responses remains uncertain, in part, due to the failure to identify the intracellular S1P receptors and the organelle(s) from which the calcium is mobilized. Interestingly, mast cells are one of the few cell types that can secrete substantial quantities of S1P upon stimulation, suggesting an important role for this metabolite in their function (13). Studies on the mast cell tumor analog, RBL-2H3, showed that cell migration to an IgE/antigen (Ag) stimulus required transactivation of S1P 1 receptors by endogenously generated S1P. Additionally, these studies demonstrated that S1P contributes to mast cell degranulation through a second receptor, S1P 2 (12).Our more recent studies demonstrated that the Src protein-tyrosine kinase (Src PTK) Lyn is required for the early phase of SphK1 activation in mast cells (14). This is mediated through the interaction of SphK1 with Lyn, thus promoting SphK1 recruitment to Fc⑀RI. Nonetheless, because SphK1 activity is delayed but not ablated by Lyn deficiency, we investigated what additional signals might be required for SphK activation. Herein, we demonstrate that both isoforms, SphK1 and SphK2, which are expressed in murine and human mast cells (HuMC), are activated upon Fc⑀RI triggering. Fyn, a second Src PTK crucial for Fc⑀RI-mediated mast cell activation (15), interacted with S...

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen–mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc∈ receptor I by modulating the balance of the counterregulatory lipids.

Numerous eukaryotic upstream promoter and enhancer regions contain a functional octamer sequence ATGCAAAT. We have examined the interactions between an octamer binding protein isolated from HeLa cells and the SV40 and immunoglobulin heavy-chain (IgH) gene enhancers. A partially purified octamer binding activity forms a single complex with the IgH enhancer octamer in a gel retardation assay, but two complexes with a SV40 enhancer fragment containing a single 72-bp element. By using point mutants and both dimethyl sulfate and diethyl pyrocarbonate modification interference assays, we show that the SV40 complexes result from binding of a factor to the octamer-related sequence ATGCAAAG (Octal) and to an adjacent previously unidentified octamer-related sequence ATGCATCT (Octa2). The base-specific interactions with Octal and Octa2 differ; chemical modifications over a 10-bp sequence TATGCAAAGC affect Octal binding whereas Octa2 binding is affected by modifications spanning a 13-bp sequence ATGCATCTCAATT in which the octamer-like sequence is not centered. The octamer binding activity has been purified extensively by a DNA affinity precipitation procedure and SDS-polyacrylamide gel electrophoresis. The purified protein, OBPI00, has an apparent molecular weight of 100 kD and binds both SV40 Octal and Octa2, as well as the IgH enhancer. The distinct interactions of OBP100 with the differently sized Octal and Octa2 binding sites suggest remarkably flexible sequence recognition between OBP100 and its binding sites.