Purification and Identification of Activating Enzymes of CS-0777, a Selective Sphingosine 1-Phosphate Receptor 1 Modulator, in Erythrocytes
| Autor: | Nobuaki Watanabe, Shin-ichi Inaba, Chizuko Yahara, Kazuishi Kubota, Masakazu Tamura, Kiyoaki Yonesu, Futoshi Nara, Tomohiro Honda, Tatsuji Matsuoka |
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| Rok vydání: | 2011 |
| Předmět: |
Proteomics
Erythrocytes Protein Purification Morpholines Immunology Sphingosine kinase Enzyme Purification Fructose Mitogen-activated protein kinase kinase Biochemistry MAP2K7 Mass Spectrometry (MS) Animals Humans Prodrugs Pyrroles Enzyme Inhibitors Phosphorylation Kinase activity Sphingosine-1-Phosphate Receptors Molecular Biology biology Cyclin-dependent kinase 2 Cyclin-dependent kinase 3 Cell Biology Amino Alcohols Protein kinase R Rats Phosphotransferases (Alcohol Group Acceptor) Receptors Lysosphingolipid HEK293 Cells Enzymology Immunosuppressor biology.protein Cyclin-dependent kinase 9 |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m110.217299 |
| Popis: | CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ∼10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug. |
| Databáze: | OpenAIRE |
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