Sphingosine 1-phosphate (S1P) receptors; S1P1 (pKi = 7.86); S1P3 (pKi = 5.93); S1P1 (pEC50 = 6.58); S1P3 (pEC50 = 7.07)

VPC23019 Is Devoid of Agonism at the S1P1 and S1P3 Receptors. VPC23019 Blocks Agonist Activity at the S1P1 and S1P3 Receptors.
The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.[1]

Determination of the Binding Constant for VPC23019 at the S1P1 and S1P3 Receptors [1]
The binding constant (Kb) for VPC23019 at the S1P1 and S1P3 receptors was determined by Schild analyses from curves that were fitted using the nonlinear regression method discussed by Lew and Angus. Briefly, nonlinear analysis of the best fit line generated by plotting the negative log of the EC50 values obtained from agonist dose-response curves, in the absence and presence of varying concentrations of antagonist, was plotted against the concentration of antagonist to give the Kb value. An F-test analysis was also performed to establish whether the antagonist did or did not meet the criteria of a simple competitive interaction. [1]

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S1P Radiolabeling[1]
[32P]S1P was prepared by incubating sphingosine and [γ-32P]ATP with cell lysate from HEK293T cells transfected transiently with human sphingosine kinase type 2 DNA. The 0.2-ml reaction contained 0.025 mm sphingosine, 1 mCi of [γ-32P]ATP (7000 Ci/mmol), and kinase buffer (10 mm Mg(C2H3O2)2 in 50 mm Tris, pH 7.5, 10 mm NaF, and 2 mm semicarbizide). The reaction was initiated by the addition of 0.02 mg of cell lysate protein and incubated at 37 °C for at least 30 min. The [32P]S1P was extracted by the addition of 1 n HCl and 2.0 m each KCl, methanol, and chloroform to the reaction mixture. The mixture was then vortexed and centrifuged at 1000 × g for 5–10 min. The organic layer was isolated, and the extraction procedure was repeated two additional times with the remaining aqueous fraction. The combined organic fractions were dried under a stream of nitrogen gas and resuspended in aqueous 0.1% fatty acid-free BSA. The specific activity of the product, [32P]S1P, is estimated to be that of the radiolabeled substrate, [γ-32P]ATP, i.e. 7000 Ci/mmol. [1]

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[32P]S1P Binding Assay[1]
Membranes containing 0.005 mg of protein from HEK293T cells transfected transiently with both receptor and G-protein DNAs were incubated in 0.5 ml of binding buffer (50 mm HEPES, 100 mm NaCl, 10 mm MgCl2, pH 7.5), 50 pm [32P]S1P, and the indicated lipid(s) for 1 h at room temperature. Bound ligand was separated from free ligand by rapid filtration and analyzed in a liquid scintillation counter. Nonspecific binding was determined as residual binding of radioligand in the presence of excess S1P to membranes, both heat-denatured and non-heat-denatured, from HEK293T cells transfected transiently with receptor and G-protein DNAs; it was typically 60% of total binding. The binding constant (Ki) associated with the ligand-receptor interaction was determined from the IC50 using the Chang-Prusoff equation (Ki = IC50/(1 + [L]/Kd). In applying this equation, the concentration of radioligand (L) is 0.05 nm and the Kd value used was that reported for the S1P-S1P1 receptor interaction, i.e. 8.1 nm. [1]

Cell Migration Assay [1]
Cell migration assays were performed using modified Boyden chambers (tissue culture-treated with a 24-mm diameter, a 0.010-mm thickness, and 0.008-mm pores, Transwell®) containing polycarbonate membranes that were coated on the underside with 0.1% gelatin. The underside of the polycarbonate membranes was rinsed once with migration medium (Dulbecco's modified Eagle's medium/F-12 without Phenol Red and 0.1% fatty acid-free BSA) and then immersed in the lower chamber containing 2 ml of migration medium. T24 cells transfected stably with human S1P1 receptor DNA were grown in Dulbecco's modified Eagle's medium/F-12 medium containing charcoal/dextran-stripped FBS and 0.010 mg/ml puromycin to 100% confluence in 150 × 25-mm tissue culture plates and serum starved for at least 12 h. Serum-starved cells were removed from culture dishes with 10× trypsin-EDTA, washed once with migration medium, and resuspended in migration medium (106 cells/ml). One milliliter of the cell suspension was added to the upper migration chamber while the S1P agonist VPC22277 (10 nm) was added to the lower chamber. Cells were allowed to migrate to the underside of the membrane for 4 h at 37 °C in the presence or absence of antagonist (VPC23019 (0–1000 nm), VPC23019, VPC23031, VPC23089, and VPC25239 (50 nm each)), which were added to the lower chamber. The migrated cells attached to the bottom surface of the membrane were removed with 10× trypsin-EDTA, their mass was determined by combining 0.1 ml of cell suspension with an equal volume of CyQuant dye solution (3.0 ml of 2× lysis buffer and 0.015 ml of CyQuant dye), and the resulting fluorescence was quantified using the FlexStation™ fluorimeter (Molecular Devices, Menlo Park, CA). Each determination represents the average of two individual migration chambers. For determination of the reversibility of the antagonism associated with VPC23019, cells were incubated with 0.01 mm VPC23019 at 37 °C for 30 min. The monolayer was washed three times with phosphate-buffered saline and processed immediately for the cell migration assay, as described above.

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Measurement of Intracellular Calcium Mobilization [1]
A FlexStation™ fluorimeter was used to measure intracellular calcium in native T24 cells and T24 cells stably transfected with either human S1P2 or human S1P3 receptor DNA. Cells were seeded (∼50,000 cells/well) in 96-well, clear bottom black microplates and left overnight at 37 °C. The cells were dye-loaded with 0.004 mm Fluo-4AM ester in a loading buffer (Hanks' balanced salt solution, pH 6.4, containing 20 mm HEPES, 0.1% fatty acid-free BSA, and 2.5 mm probenecid) for 30 min at 37 °C. After washing cell monolayers three times with phosphate-buffered saline, loading buffer was added, and the cells were exposed to sets of compounds for 3 min at 25 °C in the FlexStation™.In all cases, each concentration of every compound was tested at least in triplicate. For determination of the reversibility of the antagonism associated with VPC23019 (0.010 mm), the compound was added in combination with loading dye to the cells and incubated at 37 °C for 30 min. The cells were washed with phosphate-buffered saline and exposed to compounds immediately, as described above.

[1]. Sphingosine 1-phosphate analogs as receptor antagonists. J Biol Chem. 2005 Mar 18;280(11):9833-41. Epub 2004 Dec 8.

VPC 23019 is a secondary carboxamide resulting from the formal condensation of the carboxy group of O-phospho-D-serine with the amino group of m-octylaniline. An analogue of sphingosine-1-phosphate (S1P), it is a potent antagonist for both S1P1 and S1P3 receptors. It can inhibit S1P-induced migration of thyroid cancer cells, ovarian cancer cells, and neural stem cells. It has a role as a sphingosine-1-phosphate receptor 3 antagonist and a sphingosine-1-phosphate receptor 1 antagonist. It is a D-serine derivative, a secondary carboxamide, an organic phosphate, a phosphoric ester and an aromatic amide. It is functionally related to an O-phospho-D-serine.

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Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structure-activity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P(1) and S1P(3) receptors. The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.[1]

C17H29N2O5P

372.396325826645

372.181

C, 54.83; H, 7.85; N, 7.52; O, 21.48; P, 8.32

449173-19-7

11588811

Light yellow to yellow solid powder

4.314

4

6

12

25

429

1

CCCCCCCCC1=CC(=CC=C1)NC(=O)[C@@H](COP(=O)(O)O)N

MRUSUGVVWGNKFE-MRXNPFEDSA-N

InChI=1S/C17H29N2O5P/c1-2-3-4-5-6-7-9-14-10-8-11-15(12-14)19-17(20)16(18)13-24-25(21,22)23/h8,10-12,16H,2-7,9,13,18H2,1H3,(H,19,20)(H2,21,22,23)/t16-/m1/s1

[(2R)-2-amino-3-(3-octylanilino)-3-oxopropyl] dihydrogen phosphate

449173-19-7; VPC 23019; VPC23019; VPC-23019; (R)-2-Amino-3-((3-octylphenyl)amino)-3-oxopropyl dihydrogen phosphate; CHEMBL228102; N-(3-octylphenyl)-O-phosphono-D-serinamide; CHEBI:144948;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~335.66 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.59 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.59 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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