Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.Sphingosine 1-phosphate (S1P) 2 receptor agonists are likely to be the next generation of pharmacologic agents used to modulate immune system function. The prototype drug of this class is FTY720, which is highly efficacious in prolonging allograft survival and in ameliorating autoimmune disease in a variety of animal models (1-4). FTY720 is being tested in human trials for the indications renal transplantation and multiple sclerosis (5). Further, there is mounting evidence that S1P agonists are efficacious in animal models of atherosclerosis (6), renal ischemia-reperfusion injury (7), and acute lung injury (8).FTY720 is a sphingosine analog that, after activation by phosphorylation (to FTY720-P), disrupts lymphocyte trafficking by decreasing lymphocyte egress from lymph nodes and the thymus (9, 10). Although the precise mechanisms that underlie this phenomenon are uncertain, the profound lymphopenia that is the index of FTY720 action is dependent on agonist action at lymphocyte S1P 1 receptors. Since FTY720-P is also a potent agonist at the S1P 3 , S1P 4 , and S1P 5 receptors (11, 12), it remains unknown whether the multiple therapeutic benefits of the drug correlate with agonist activity at the S1P 1 receptor. The propensity for S1P 1 receptor responses to desensitize (13) and the similar behaviors of S1P 1 receptor null thymocytes and FTY720-treated mouse lymphocytes have led to the suggestion that FTY720-P is a functional antagonist (14). In this scenario, the drug exaggerates S1P tone to the extent that the lymphocyte S1P 1 receptor signaling is chronically down-regulated.The kinase(s) responsible for FTY720 activation is the gateway whereby S1P signaling can be accessed readily with a therapeutic agent. Knowledge of this enzyme is important specifically to guide S1P prodrug design and generally to gain insight into the normal role of S1P in immune function. The identity of the kinase is not known currently; two candidates are sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These enzymes, which are expressed widely, catalyz...

Compound 1 (FTY720, Fingolimod) represents a new generation of immunosuppressant that modulates lymphocyte trafficking by interacting with the S1P 1 receptor. Compound 1 also provides a template molecule for studying the molecular biology of S1P receptors and related enzymes (kinases and phosphatases). In this study, two conformationally constrained analogues of 1 (3a and 3c) were asymmetrically synthesized in high optical purity. In vitro assessment documented that both analogues are Sphk2 substrates, their phosphorylated species are potent S1P 1 receptor agonists, and 3a-P is a potent S1P 3 antagonist. After oral administration in mice, both compounds evoked lymphopenia, but their duration of action differed markedly.

Trovafloxacin (Trovan) is a fluoroquinolone antibiotic drug with a long half-life and broad-spectrum activity. Since its entry into the market in 1998, trovafloxacin has been associated with numerous cases of hepatotoxicity, which has limited its clinical usefulness. Trovafloxacin possesses two substructural elements that have the potential to generate reactive intermediates: a cyclopropylamine moiety and a difluoroanilino system. The results presented here describe the in vitro metabolic activation of a synthetic drug model (DM) of trovafloxacin that contains the cyclopropylamine moiety. Cyclopropylamine can be oxidized to reactive ring-opened products-a carbon-centered radical and a subsequently oxidized alpha,beta-unsaturated aldehyde. Experiments with monoamine oxygenases, horseradish peroxidase, flavin monooxygenase 3, and cDNA-expressed P450 isoenzymes revealed that P450 1A2 oxidizes DM to a reactive alpha,beta-unsaturated aldehyde, M 1. Furthermore, myeloperoxidase (MPO) was also demonstrated to oxidize DM in the presence of chloride ion to produce M 1. DM proved to be a suicide inhibitor of MPO while showing no inhibition of P450 1A2. The structure of the reactive metabolite was confirmed by LC-MS/MS analysis by comparison with a synthetic standard. M 1 was further shown to react with glutathione and the related thiol nucleophile, 4-bromobenzyl mercaptan, suggesting the potential of this intermediate to react with protein nucleophiles. In summary, these data provide evidence that trovafloxacin-induced hepatotoxicity may be mediated through the oxidation of the cyclopropylamine substructure to reactive intermediates that may form covalent adducts to hepatic proteins, resulting in damage to liver tissue.

Sphingosine 1-phosphate (S1P) is a phospholipid that binds to a set of G protein-coupled receptors (S1P 1 -S1P 5 ) to initiate an array of signaling cascades that affect cell survival, differentiation, proliferation, and migration. On a larger physiological scale, the effects of S1P on immune cell trafficking, vascular barrier integrity, angiogenesis, and heart rate have also been observed. An impetus for the characterization of S1P-initiated signaling effects came with the discovery that FTY720 [fingolimod; 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] modulates the immune system by acting as an agonist at S1P 1 . In the course of structureactivity relationship studies to better understand the functional chemical space around FTY720, we discovered conformationally constrained FTY720 analogs that behave as S1P receptor typeselective antagonists. Here, we present a pharmacological profile of a lead S1P 1/3 antagonist prodrug, 1-(hydroxymethyl)-3-(3-octylphenyl)cyclobutane (VPC03090). VPC03090 is phosphorylated by sphingosine kinase 2 to form the competitive antagonist species 3-(3-octylphenyl)-1-(phosphonooxymethyl)cyclobutane (VPC03090-P) as observed in guanosine 5Ј-O-(3-[ 35 S]thio)triphosphate binding assays, with effects on downstream S1P receptor signaling confirmed by Western blot and calcium mobilization assays. Oral dosing of VPC03090 results in an approximate 1:1 phosphorylated/alcohol species ratio with a half-life of 30 h in mice. Because aberrant S1P signaling has been implicated in carcinogenesis, we applied VPC03090 in an immunocompetent mouse mammary cancer model to assess its antineoplastic potential. Treatment with VPC03090 significantly inhibited the growth of 4T1 primary tumors in mice. This result calls to attention the value of S1P receptor antagonists as not only research tools but also potential therapeutic agents.