PF-543

Catalog No.S7177

PF-543 Chemical Structure

Molecular Weight(MW): 465.6

PF-543, 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.

Size Price Stock Quantity  
In DMSO USD 120 In stock
USD 97 In stock
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1 Customer Review

  • PF-543 is anti-proliferative and cytotoxic to CRC cells. Human CRC cell lines, including HCT-116 (a-d), DLD-1 and HT-29 (e), as well as patient-derived primary colon cancer cells (P1, P2 and P3, f), were incubated with regular or PF-543-containg medium for indicated time, cell proliferation was tested by viable cell counting assay (a) and colony formation assay (b); Cell survival was examined by MTT assay (c-f). Expression of SphK1 and Tubulin (the equal loading) was examined by Western blots (e and f, upper panels). Relative SphK1 expression (verse Tubulin) was quantified. “C” stands for untreated control cells (For all Figs). Values represented the mean ± SEM (n = 5). Experiments in this figure were repeated three times, with similar results achieved. *P < 0.05 versus “C” group.

    Biochem Biophys Res Commun, 2016, 470(3):728-34.. PF-543 purchased from Selleck.

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Biological Activity

Description PF-543, 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.
Features The most potent inhibitor of SphK1 described to date.
Targets
SphK1 [1] SphK1 [1]
2.0 nM 3.6 nM(Ki)
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]

Protocol

Kinase Assay:

[1]

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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:

[1]

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  • Cell lines: 1483, A549, LN229, Jurkat, U937, MCF-7
  • Concentrations: ~1 μM
  • Incubation Time: 7 days
  • Method:

    CellTiter-Glo Assay


    (Only for Reference)

Solubility (25°C)

In vitro DMSO 93 mg/mL (199.74 mM)
Ethanol 93 mg/mL (199.74 mM)
Water slightly soluble or insoluble
In vivo Add solvents individually and in order:

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 465.6
Formula

C27H31NO4S

CAS No. 1415562-82-1
Storage powder
in solvent
Synonyms N/A

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Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID