cs-0777 and 1-deoxy-1-morpholinofructose

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes. 2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 μM to 1060 μM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate. 3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 μM to 32.1 μM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes. 4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

Topics: Alkaline Phosphatase; Amino Alcohols; Enzyme Inhibitors; Erythrocytes; Female; Fructose; Humans; Intestine, Small; Kidney; Kinetics; Levamisole; Male; Microsomes, Liver; Morpholines; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyrroles; Receptors, Lysosphingolipid; Recombinant Proteins

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.

Topics: Amino Alcohols; Animals; Enzyme Inhibitors; Erythrocytes; Fructose; HEK293 Cells; Humans; Morpholines; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Pyrroles; Rats; Receptors, Lysosphingolipid; Sphingosine-1-Phosphate Receptors