PF-543 1415562-82-1_S1P Receptor_GPCR & G Protein_Signaling Pathways_Products

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.

Physicochemical Properties

Molecular Formula

C27H31NO4S

Molecular Weight

465.6

Exact Mass

465.197

Elemental Analysis

C, 69.65; H, 6.71; N, 3.01; O, 13.74; S, 6.89

CAS #

1415562-82-1

Related CAS #

PF-543 Citrate; 1415562-83-2; PF-543 hydrochloride; 1706522-79-3

PubChem CID

66577038

Appearance

White to off-white Waxy semisolid

Density

1.2±0.1 g/cm3

Boiling Point

666.0±55.0 °C at 760 mmHg

Flash Point

356.6±31.5 °C

Vapour Pressure

0.0±2.1 mmHg at 25°C

Index of Refraction

1.610

LogP

4.16

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

679

Defined Atom Stereocenter Count

SMILES

CC1=CC(OCC2=CC=C(CN3[C@@H](CO)CCC3)C=C2)=CC(CS(C4=CC=CC=C4)(=O)=O)=C1

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

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Shipping Condition

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

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]. Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1. Biochem J. 2012 May 15;444(1):79-88.

[2]. Effect of the sphingosine kinase 1 selective inhibitor, PF-543 on arterial and cardiac remodelling in a hypoxic model of pulmonary arterial hypertension. Cell Signal. 2016 Aug;28(8):946-55.

[3]. Induction of autophagy by sphingosine kinase 1 inhibitor PF-543 in head and neck squamous cell carcinoma cells. Cell Death Discov. 2017 Aug 14;3:17047.

Additional Infomation

[(2R)-1-[[4-[[3-(benzenesulfonylmethyl)-5-methylphenoxy]methyl]phenyl]methyl]-2-pyrrolidinyl]methanol is a sulfonamide.

Solubility Data

Solubility (In Vitro)

DMSO: 93~100 mg/mL (199.7~214.8 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 5 mg/mL (10.74 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

Solubility in Formulation 2: ≥ 5 mg/mL (10.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

(Please use freshly prepared in vivo formulations for optimal results.)

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.

PeptideDB

PF-543 1415562-82-1

CAS No.: 1415562-82-1

Physicochemical Properties

Biological Activity

Solubility Data