Purification and Identification of Activating Enzymes of CS-0777, a Selective Sphingosine 1-Phosphate Receptor 1 Modulator, in Erythrocytes

Abstract

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.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS‐917

“…This is not an uncommon observation as we reported previously (Yonesu et al. ) and as written by others (Ryan ), indicating a presence of inhibitors of the enzyme reaction in crude samples. With regard to identification strategy, CES1 and CTSA can be expected as a CS‐917 esterase, ELA3B could be speculated based on the activity of ELA2 for PSI‐7851 (Murakami et al.…”

“…In the purification scheme, recovery of enzyme activities at the early purification steps showed more than 100%. This is not an uncommon observation as we reported previously (Yonesu et al 2011) and as written by others (Ryan 1996), indicating a presence of inhibitors of the enzyme reaction in crude samples. With regard to identification strategy, CES1 and CTSA can be expected as a CS-917 esterase, ELA3B could be speculated based on the activity of ELA2 for PSI-7851 (Murakami et al 2010), however identification of SMPDL3A as an R-134450 phosphoramidase was completely unexpected because SMPDL3A was very far from the HINT family proteins which have only been reported as having phosphoramidase activity.…”

Identification of a Metabolizing Enzyme in Human Kidney by Proteomic Correlation Profiling

“…Given this observation, it is intriguing that identified kinases for fingolimod and CS-0777 were also different (6, 28). That different enzymes are responsible for catalyzing phosphorylation and de-phosphorylation of these related molecules could have important implications for drug metabolism, as each could thus be subject to distinct pharmacological effects ( e.g.…”

“…Protein identification by mass spectrometry subsequently revolutionized this technology by enabling identification of proteins at much lower abundances: individual proteins could then be associated with specific activities as soon as a band in SDS-PAGE could be observed, even when the purified protein was far from homogeneity (4–6). Although this streamlined the workflow by reducing the required starting materials as well as the separation steps for protein purification, a faster and more generalized approach from smaller starting material has still been desired because some proteins are physiochemically difficult for example in solubilization and stability.…”

“…Sphingosine kinase 2 (SPHK2) was identified as the major kinase of fingolimod (21, 28, 29) and lipid phosphate phosphatase 3 (LPP3) was reported to be a phosphatase for fingolimod-P dephosphorylation (30), although contribution of LPP3 in vivo has not been fully studied. In our previous work, we have identified CS-0777 kinases in human blood as fructosamine 3-kinase-related protein (FN3K-RP) and fructosamine 3-kinase (FN3K) (6), whereas the phosphatase of CS-0777-P had not been identified thus far.…”