Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1

Abstract

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

A real-time high-throughput fluorescence assay for sphingosine kinases

“…4B , inset) revealed an IC 50 value of 16 nM. A previous study using 1 µM FITC-Sph as a substrate reported an IC 50 of 3.6 nM ( 19 ). SphK1 inhibition kinetics with DMS revealed an IC 50 of 15.2 µM ( Fig.…”

“…However, the signal is too low to accurately measure basal SphK activity. Importantly, overexpressed SphK1 activity was strongly inhibited by PF-543 ( 19 ), showing specifi city to a SphK1 catalyzed reaction ( Fig. 2H ).…”

“…As a fi rst approach, we examined SphK1 activity in the presence of the potent SphK1 inhibitor PF-543 (250 nM) ( 19 ), which almost completely suppressed the ATP/SphK1-dependent appearance of the 550 nm peak ( Fig. 4A ).…”

Palmitate and Lipopolysaccharide Trigger Synergistic Ceramide Production in Primary Macrophages

“…To determine the impact of LPS stimulation on sphingolipid content of primary macrophages cultured for 16 h in the presence of 500 M palmitate, we used LC-MS/MS to examine metabolites in this pathway (24). As seen in Table 1 and Fig.…”

Sphingolipid in Lung Cancer Pathogenesis and Therapy

“…Although SphK1 is elevated in lung cancer patients suggesting a potentially important role for this enzyme in lung tumor cell proliferation and survival [43,53], results with novel, highly potent, and selective inhibitors to SphK1 in tumor cells did not affect their growth in vitro or in vivo, suggesting that tumor SphK1 may not be an efficacious therapeutic target for cancer [54,55]. Hence, inhibitors of SphK2 are developed and tested for inducing lung cancer cell death, among which ABC294640 is a first-in-class drug [56].…”