A rapid assay for assessment of sphingosine kinase inhibitors and substrates

Yugesh Kharel, Thomas P. Mathews, Andrew J. Kennedy, Joseph D. Houck, Timothy L. MacDonald, Kevin R. Lynch

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-32P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, Km, and the Ki values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.

Original languageEnglish (US)
Pages (from-to)230-235
Number of pages6
JournalAnalytical biochemistry
Issue number2
StatePublished - Apr 15 2011
Externally publishedYes


  • High-throughput assay
  • Sphingosine 1-phosphate
  • Sphingosine kinase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'A rapid assay for assessment of sphingosine kinase inhibitors and substrates'. Together they form a unique fingerprint.

Cite this