PF-543 (Sphingosine Kinase 1 Inhibitor II), a novel sphingosine-competitive inhibitor of SphK1, inhibits SphK1 with IC50 and Ki of 2.0 nM and 3.6 nM.

SPHK1:2.0 nM, SPHK1:3.6 nM(Ki)

FITC-S1P quantification/Caliper assay: A 384-well format of the SphK enzyme assay based on separation of FITC-S1P from unreacted FITC-sphingosine substrate using a microfluidic capillary electrophoresis mobility-shift system is developed. Briefly, 3 nM SphK1–His6 is incubated with 1 μM FITC-sphingosine, 20 μM ATP and 10 μM compound (a final concentration of DMSO of 2 %) in a buffer containing 100 mM Hepes (pH 7.4), 1 mM MgCl2,0.01% Triton X-100, 10% glycerol, 100 μM sodium orthovanadate and 1 mM DTT for 1 h in a 384-well Matrical MP-101-1-PP plate. Reaction mixtures (10 μL) are quenched by the addition of 20 μL of 30 mM EDTA and 0.15% Coating Reagent-3 in 100 mM Hepes, and a small aliquot of each reaction (a few nanolitres) is analysed in the Caliper LabChip 3000 instrument under -1.5 psi (psi=6.9 kPa) pressure, a downstream voltage of -1900 V and a sip time of 0.2 s. Phosphorylated fluorescent product and unphosphorylated fluorescent substrate appeared as distinctive peaks and are quantified using the Caliper data.

CellTiter-Glo Assay (Only for Reference)

Sphingosine Kinase 1 Inhibitor II, PF 543

H2O: <1 mg/mL

DMSO: 93 mg/mL (199.74 mM), Sonication is recommended.

Ethanol: 93 mg/mL (199.74 mM), Sonication is recommended.

10% DMSO+90% Corn Oil: 5 mg/mL (10.74 mM), Sonication is recommended.