Development of a Simple and Robust Assay to Screen for Inhibitors of Sphingosine Kinases

Abstract

Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), which plays important roles in a wide variety of intra- and extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and autoradiography to detect the product S1P. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC. The assay is based on ³³P-phosphate transfer from [γ-³³P]ATP to sphingosine and subsequent detection of the [³³P]S1P using AquaBind™ plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K _m values for D-erythro-sphingosine and ATP were determined to be 17.5 μM and 19.2 μM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 μM and 450 μM, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by a high signal-to-background ratio, small variation, and excellent Z factors.