PF-543 hydrochloride

PF543 is a cell-permeable hydroxyl methylpyrrolidine compound that inhibits SphK-1/SphK1-catalyzed sphingosine phosphorylation in a reversible and sphingosine-competitive manner, exhibiting no affinity toward S1P receptors and much reduced inhibitory activity against Sphk2 (6.8% inhibition at 10 μM) or 46 other lipid and portein kinases (IC50 >10 μM). In the SphK1-overexpression 1483 head and neck carcinoma cells, PF-543 decreases the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. PF-543 binds SphK1 reversibly (k _off t1/2=8.5 min) and with high affinity and the binding constant (K_d) is 5 nM. PF543 had no effect on the proliferation and survival of 1483, A549, LN229, Jurkat, U937 and MCF-7 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 is effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. ^[1]

PF-543, a potent sphingosine kinase 1 inhibitor, reduced dysfunctional hypertrophy, associated with protection against cardiomyocyte apoptosis.

• 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.

• Cell lines

  1483, A549, LN229, Jurkat, U937, MCF-7

• Concentrations

  ~1 μM

• Incubation Time

  7 days

• Method

  CellTiter-Glo Assay

• Animal Models

  C57BL/6 mice

• Dosages

  1 mg/kg

• Administration

  i.p.