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

Area of Science:

• Biochemistry
• Enzymology
• Cellular Signaling

Background:

• Sphingosine kinases (SphKs), comprising SphK1 and SphK2, are crucial enzymes regulating cellular processes.
• Existing methods for SphK activity assessment often require complex procedures, radioactive materials, or specialized equipment.

Purpose of the Study:

• To develop a novel, simplified assay for real-time monitoring of SphK1 and SphK2 activity.
• To enable efficient characterization of SphK inhibitors and mutants, and facilitate high-throughput screening.

Main Methods:

• Development of a fluorescence-based assay utilizing 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph).
• Real-time monitoring of SphK-dependent changes in NBD-Sph fluorescence.
• Assay performed in 384-well plate format with small reaction volumes.

Main Results:

• The assay demonstrated dose-proportional responses to enzyme, substrate, and inhibitor concentrations.
• Determined binding affinities for NBD-Sph and IC50 values for inhibitors were consistent with established methods.
• The assay is versatile, simple, and suitable for routine use and high-throughput screening.

Conclusions:

• The developed fluorescence assay provides a robust and convenient method for assessing SphK activity.
• This assay facilitates the characterization of SphK modulators and mutants, and is readily adaptable for compound library screening.