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

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive Materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to Enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with Other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.