PF-543

Alias: PF543; PF-543 HCl;PF543; PF 543; PF-543 hydrochloride
Cat No.:V1504 Purity: ≥98%
PF-543 (also called PF543; PF 543; Sphingosine Kinase 1 Inhibitor II) is a novel cell-permeable and sphingosine-competitive inhibitor of SphK1 with potential antitumor activity.
PF-543 Chemical Structure CAS No.: 1415562-82-1
Product category: S1P Receptor
This product is for research use only, not for human use. We do not sell to patients.
Size Price Stock Qty
5mg
10mg
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Other Forms of PF-543:

  • PF-543 Citrate
  • PF-543 HCl
Official Supplier of:
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

PF-543 (also called PF543; PF 543; Sphingosine Kinase 1 Inhibitor II) is a novel cell-permeable and sphingosine-competitive inhibitor of SphK1 with potential antitumor activity. It shows >100-fold selectivity for SphK1 over the SphK2 isoform and inhibits SphK1 with IC50s and Ki of 2.0 nM and 3.6 nM.

Biological Activity I Assay Protocols (From Reference)
Targets
SphK1
ln Vitro

In vitro activity: PF543, a cell-permeable hydroxyl methylpyrrolidine compound, exhibits no affinity toward S1P receptors and much reduced inhibitory activity against 46 other lipid and portein kinases (IC50 >10 μM) and Sphk2 (6.8% inhibition at 10 μM) that is catalyzed by SphK-1/SphK1 in sphingosine phosphorylation. PF-543 reduces endogenous S1P levels in the SphK1-overexpressing 1483 head and neck carcinoma cells by ten times while increasing sphingosine levels in proportion. PF-543 exhibits strong affinity and reversible binding of SphK1 (k off t1/2=8.5 min), with a binding constant (Kd) of 5 nM. Despite a significant alteration in the cellular S1P/sphingosine ratio, PF543 had no effect on the survival and proliferation of 1483, A549, LN229, Jurkat, U937, and MCF-7 cells. Since PF-543 is a potent inhibitor of S1P formation in whole blood, it can be concluded that the primary source of S1P in human blood is the SphK1 isoform of sphingosine kinase.

ln Vivo
In human whole blood, the inhibitory activity of PF-543 was also investigated ex vivo. The preparation of human whole blood with elevated SphK1 activity allowed for the prompt conversion of C17-sphingosine to C17-S1P. Treatment with PF-543 demonstrated that the compound was a strong SphK1 inhibitor, able to prevent >90% of C17-S1P formation.
Enzyme Assay
The SphK enzyme assay, which uses a microfluidic capillary electrophoresis mobility-shift system to separate unreacted FITC-sphingosine substrate from FITC-S1P, is developed in a 384-well format. In summary, 1 μM FITC-sphingosine, 20 μM ATP, and 10 μM compound (with a final DMSO concentration of 2%), along with 3 nM SphK1–His6, are incubated for 1 hour in a 384-well Matrical MP-101-1-PP plate with a buffer that contains 100 mM Hepes (pH 7.4), 1 mM MgCl2, 0.01% Triton X-100, 10% glycerol, 100 μM sodium orthovanadate, and 1 mM DTT. The reaction mixtures (10 μL) are quenched by adding 20 μL of 30 mM EDTA and 0.15% Coating Reagent-3 in 100 mM Hepes. A small aliquot of each reaction, a few nanoliters, is then analyzed in the Caliper LabChip 3000 instrument at a downstream voltage of -1900 V, a sip time of 0.2 s, and a pressure of -1.5 psi (psi=6.9 kPa). The Caliper data are used to quantify the distinct peaks that appeared as phosphorylated fluorescent product and unphosphorylated fluorescent substrate.
Cell Assay
In 1483 head and neck cancer cell cultures that expressed high levels of SphK1 and produced S1P at an exceptionally high rate, pretreatment with PF-543 for one hour reduced endogenous S1P levels by ten times while increasing sphingosine levels proportionately. Significant inhibition of SphK1 BY pf-543 was observed. Specific inhibition of SphK1, however, did not affect the survival and proliferation of 1483 cell cultures, even though the cellular S1P/sphingosine rate changed dramatically.
Animal Protocol
Dissolved in vehicle (PBS); 0 or 30 mg/kg; injected via the tail vein
Mice: RB-005 or PF-543 (10 or 30 mg/kg) dissolved in vehicle (PBS) are injected intraperitoneally (IV) into two-month-old female C57BL/6 J mice. Following the administration of the drug, tail vein bleeds are used to extract 20 μL of blood at intervals of 15, 30, 1, 1, 4, 6, and 24 hours. Using MS analysis, drug concentration is ascertained.
References

[1]. Biochem J . 2012 May 15;444(1):79-88.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C27H31NO4S
Molecular Weight
465.6
Exact Mass
465.2
Elemental Analysis
C, 69.65; H, 6.71; N, 3.01; O, 13.74; S, 6.89
CAS #
1415562-82-1
Appearance
Waxy semisolid
SMILES
CC1=CC(=CC(=C1)OCC2=CC=C(C=C2)CN3CCC[C@@H]3CO)CS(=O)(=O)C4=CC=CC=C4
InChi Key
NPUXORBZRBIOMQ-RUZDIDTESA-N
InChi Code
InChI=1S/C27H31NO4S/c1-21-14-24(20-33(30,31)27-7-3-2-4-8-27)16-26(15-21)32-19-23-11-9-22(10-12-23)17-28-13-5-6-25(28)18-29/h2-4,7-12,14-16,25,29H,5-6,13,17-20H2,1H3/t25-/m1/s1
Chemical Name
[(2R)-1-[[4-[[3-(benzenesulfonylmethyl)-5-methylphenoxy]methyl]phenyl]methyl]pyrrolidin-2-yl]methanol
Synonyms
PF543; PF-543 HCl;PF543; PF 543; PF-543 hydrochloride
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO: 93~100 mg/mL (199.7~214.8 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.1478 mL 10.7388 mL 21.4777 mL
5 mM 0.4296 mL 2.1478 mL 4.2955 mL
10 mM 0.2148 mL 1.0739 mL 2.1478 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

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