Home Immunology & Inflammation SPHK inhibitor - Autophagy activator - Apoptosis related activator PF-543 hydrochloride For research use only.

PF-543 hydrochloride

PF-543 hydrochloride, a novel sphingosine-competitive inhibitor of SphK1, inhibits SphK1 with IC50 and Ki of 2.0 nM and 3.6 nM, exhibits >100-fold selectivity over the SphK2 isoform. PF-543 hydrochloride induces apoptosis, necrosis, and autophagy.

PF-543 hydrochloride Chemical Structure

PF-543 hydrochloride Chemical Structure

CAS No. 1706522-79-3

Purity & Quality Control

PF-543 hydrochloride Related Products

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human PAMSC cells Function assay 100 nM 24 h Decrease of SK1 expression in human PAMSC cells at 100 nM after 24 hrs by Western blot analysis 24396570
Sf9 cells Function assay Inhibition of human recombinant SphK1 expressed in Sf9 cells assessed as radiolabeled products using 5 uM sphingosine and 10 uM gamma[32P]ATP by liquid scintillation counting 25643074
Sf21 cells Function assay 90 mins Inhibition of His6 tagged human SphK1 expressed in Sf21 cells using FITC-sphingosine as substrate preincubated for 90 mins prior substrate addition by Caliper assay, Ki=4.3 nM 25516793
PC3 Antiproliferative assay 72 hrs Antiproliferative activity against human PC3 cells after 72 hrs by MTS assay, GI50=19μM. 26780304
HEK293 Function assay 10 uM 16 hrs Induction of SK1 degradation in HEK293 cells at 10 uM after 16 hrs by western blotting analysis in presence of proteasome inhibitor MG132 26780304
MDA1483 Function assay 30 mins Inhibition of SPHK1 in human MDA1483 cells using C17-sphingosine as substrate assessed as reduction in C17-S1P formation preincubated for 30 mins with substrate measured after 15 mins by LC-MS analysis, IC50=0.0008μM. 28231433
Sf9 Function assay 15 mins Inhibition of human C-terminal His6-tagged SPHK1 expressed in fall armyworm sf9 cells assessed as reduction in ADP formation using sphingosine as substrate preincubated for 15 mins followed by substrate addition after 1 hr by transcreener-based fluorescen, IC50=0.002μM. 28231433
Sf9 Function assay 1 hr Inhibition of human C-terminal His6-tagged SPHK1 expressed in fall armyworm sf9 cells assessed as reduction in S1P formation using sphingosine as substrate after 1 hr by FITC-based caliper assay, IC50=0.0027μM. 28231433
Sf21 Function assay 1 hr Inhibition of recombinant human C-terminal His-tagged SPHK1 expressed in fall armyworm sf21 cells using sphingosine as substrate after 1 hr by FITC-based caliper assay, Ki=0.0036μM. 28406646
Sf21 Function assay 1 hr Inhibition of recombinant C-terminal His6-tagged human SphK1 expressed in baculovirus infected sf21 cells using FITC-sphingosine as substrate after 1 hr in presence of ATP by microfluidic capillary electrophoresis mobility shift assay, IC50=0.0027μM. 28408190
Sf21 Function assay 1 hr Inhibition of human recombinant C-terminal His6 tagged SphK2 expressed in baculovirus infected sf21 cells using FITC-sphingosine as substrate after 1 hr in presence of ATP by microfluidic capillary electrophoresis mobility shift assay, IC50=0.36μM. 28408190
Sf21 Function assay Inhibition of human C-terminal His6-tagged SphK1 expressed in baculovirus infected Sf21 insect cells, Ki=0.0036μM. 28822281
HEK293 Function assay 30 mins Inhibition of GFP-tagged SK1 (unknown origin) expressed in HEK293 cells using Sph as substrate measured after 30 mins in presence of [gamma32P]ATP by Cerenkov counting analysis, IC50=0.028μM. 30889352
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Biological Activity

Description PF-543 hydrochloride, a novel sphingosine-competitive inhibitor of SphK1, inhibits SphK1 with IC50 and Ki of 2.0 nM and 3.6 nM, exhibits >100-fold selectivity over the SphK2 isoform. PF-543 hydrochloride induces apoptosis, necrosis, and autophagy.
Features The most potent inhibitor of SphK1 described to date.
Targets
SphK1 [1]
(Cell-free assay)		 SphK1 [1]
(Cell-free assay)
2.0 nM 3.6 nM(Ki)
In vitro
In vitro

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. [1]
Kinase Assay 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 Research Cell lines 1483, A549, LN229, Jurkat, U937, MCF-7
Concentrations ~1 μM
Incubation Time 7 days
Method

CellTiter-Glo Assay
In Vivo
In vivo

PF-543, a potent sphingosine kinase 1 inhibitor, reduced dysfunctional hypertrophy, associated with protection against cardiomyocyte apoptosis.
Animal Research Animal Models C57BL/6 mice
Dosages 1 mg/kg
Administration i.p.

Chemical Information & Solubility

Molecular Weight 502.07 Formula

C27H32ClNO4S
CAS No. 1706522-79-3 SDF Download PF-543 hydrochloride SDF
Smiles CC1=CC(=CC(=C1)OCC2=CC=C(C=C2)CN3CCCC3CO)CS(=O)(=O)C4=CC=CC=C4.Cl
Storage (From the date of receipt)

In vitro
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DMSO : 100 mg/mL ( (199.17 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 100 mg/mL

Water : Insoluble


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