product name PF-543
Description: PF-543 is a novel sphingosine-competitive inhibitor of SphK1 that inhibits SphK1 with IC50 and Ki of 2.0 nM and 3.6 nM, it exhibits >100-fold selectivity over the SphK2 isoform. PF-543 was 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. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.
References: Biochem J. 2012 May 15;444(1):79-88.
465.6
Formula
C27H31NO4S
CAS No.
1415562-82-1
Storage
-20℃ for 3 years in powder form
-80℃ for 2 years in solvent
Solubility (In vitro)
DMSO: 93 mg/mL (199.7 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Solubility (In vivo)
Synonyms
other peoduct :
| In Vitro |
In vitro activity: 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 (Kd) 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. Kinase 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 Assay: In 1483 head and neck carcinoma cell cultures expressing high levels of SphK1 and with an unusually high rate of S1P production, pretreatment of PF-543 for 1 hr decreased the level of endogenous S1P by 10-fold with a proportional increase in the level of sphingosine. It indicated a significant inhibition of SphK1 BY pf-543. However, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cell cultures, despite a dramatic change in the cellular S1P/sphingosine rate. |
|---|---|
| In Vivo | The inhibitory activity of PF-543 was also examined ex vivo in human whole blood. Human whole blood with high SphK1 activity was prepared, which was able to quickly convert C17-sphingosine to C17-S1P. PF-543 treatment showed that PF-543 was a potent inhibitor of SphK1, capable of blocking >90% C17-S1P formation |
| Animal model | |
| Formulation & Dosage | |
| References | Biochem J. 2012 May 15;444(1):79-88. |
