Development of a simple and robust assay to screen for inhibitors of sphingosine kinases.
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 (33)P-phosphate transfer from [gamma-(33)P]ATP to sphingosine and subsequent detection of the [(33)P]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 microM and 19.2 microM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 microM and 450 microM, 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.
Togame
Hiroko
H
Discovery Research Laboratories, Shionogi & Co., Ltd., Sagisu, Osaka, Japan. hiroko.togame@shionogi.co.jp
Dodo
Reiko
R
Inaoka
Tetsuya
T
Reinemer
Peter
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D001331:Automation; D004353:Drug Evaluation, Preclinical; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099157:Sphingosine Kinase
D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D011994:Recombinant Proteins; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate