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)

PF543是一种可渗透细胞的羟甲基吡咯烷化合物，以可逆及与鞘氨醇竞争的方式抑制SphK-1/SphK1催化的鞘氨醇磷酸化，对S1P受体没有亲和力，并且对Sphk2（10 μM时抑制率为6.8%）或其他46种脂质和蛋白激酶的抑制活性大大减弱（IC50 >10 μM）。在过表达SphK1的1483头颈癌细胞中，PF-543将内源性S1P水平降低10倍，同时鞘氨醇水平成比例增加。PF-543以高亲和力可逆地结合SphK1（解离半衰期k off t1/2=8.5分钟），结合常数（Kd）为5 nM。PF543对1483、A549、LN229、Jurkat、U937和MCF-7细胞的增殖和存活无影响，尽管细胞内S1P/鞘氨醇比例发生了显著变化。PF-543作为全血中S1P形成的强效抑制剂有效，表明鞘氨醇激酶的SphK1亚型是人血液中S1P的主要来源。[1]

在小鼠低氧模型的肺动脉高压研究中，强效的sphingosine kinase 1抑制剂PF-543的给药并不影响血管重塑，但能减少右心室肥大。向小鼠给予10 mg/kg的PF-543并持续24小时，可导致肺血管中SK1表达的下降[2]。

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)