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BioAssay: AID 540367

Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 2 (S1P2) agonist assay

Name: Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 2 (S1P2) agonist assay ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 540367
Data Source: The Scripps Research Institute Molecular Screening Center (S1P2_AG_BLA_384_1X%INH)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-08-19
Hold-until Date: 2012-03-01
Modify Date: 2012-03-01

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
373S1P3 Agonist Primary HTS and Confirmation AssaysscreeningPrimary screen (S1P3 agonists in singlicate)
439S1P3 Agonist Dose-Response Potency AssayconfirmatoryDose response (S1P3 agonists in triplicate)
1192Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Purchased AnaloguesconfirmatoryDose response (S1P3 agonists in triplicate)
540309Summary of probe development efforts to identify agonists of Sphingosine 1-Phosphate Receptor 3 (S1P3)summarySummary (S1P3 agonists in triplicate)
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Oldstone, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: U01 AI074564
Grant Proposal PI: Michael Oldstone, TSRI

External Assay ID: S1P2_AG_BLA_384_1X%INH

Name: Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 2 (S1P2) agonist assay


Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions in both intracellular (1) and extracellular compartments (2), including a wide variety of physiological responses such as heart rate (3-4), coronary artery caliber, endothelial integrity, and lymphocyte recirculation (4-7). These responses are mediated through high-affinity interactions with five members of the endothelial differentiation gene (EDG) family of plasma membrane-localized G-protein-coupled receptors (GPCRs), the sphingosine lipid receptors, S1P1-5 (8-10). S1P3 receptor couples promiscuously to Gi, Gq, and G12/13 proteins (11-13). Its expression is widespread (14-16). The S1P3 knockout mouse is phenotypically normal (14). Most S1P-mediated responses on endothelial cells occur via the S1P1 receptor alone or in combination with the S1P3 receptor. Bradycardia and hypertension are clearly associated with S1P3 activation and its expression patterns in cardiac tissue (3, 17). The use of the S1P1-selective agonist SEW2871 together with S1P3-deletant mice showed that activation of S1P3 regulates sinus rhythm, whereas activation of S1P1 plays no discernable role in the process (4). S1P3 on dendritic cells has been identified as a major exacerbating factor for mortality during sepsis by playing a role in the critical linkage of inflammation and coagulation pathways downstream of the thrombin cascade (18). A potent and selective S1P3 agonist would be useful in dissecting the complexities of S1P-mediated physiological processes in which S1P3 is involved, including bradycardia and hypertension.


1. Goetzl, E. J., Wang, W., McGiffert, C., Liao, J. J., and Huang, M. C. (2007) Sphingosine 1-phosphate as an intracellular messenger and extracellular mediator in immunity, Acta Paediatr Suppl 96, 49-52
2. Spiegel, S., and Milstien, S. (2003) Sphingosine-1-phosphate: an enigmatic signalling lipid, Nat Rev Mol Cell Biol 4, 397-407.
3. Forrest, M., Sun, S. Y., Hajdu, R., Bergstrom, J., Card, D., Doherty, G., Hale, J., Keohane, C., Meyers, C., Milligan, J., Mills, S., Nomura, N., Rosen, H., Rosenbach, M., Shei, G. J., Singer, II, Tian, M., West, S., White, V., Xie, J., Proia, R. L., and Mandala, S. (2004) Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes, J Pharmacol Exp Ther 309, 758-768.
4. Sanna, M. G., Liao, J., Jo, E., Alfonso, C., Ahn, M. Y., Peterson, M. S., Webb, B., Lefebvre, S., Chun, J., Gray, N., and Rosen, H. (2004) Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate, J Biol Chem 279, 13839-13848.
5. Alfonso, C., McHeyzer-Williams, M. G., and Rosen, H. (2006) CD69 down-modulation and inhibition of thymic egress by short- and long-term selective chemical agonism of sphingosine 1-phosphate receptors, Eur J Immunol 36, 149-159.
6. Jo, E., Sanna, M. G., Gonzalez-Cabrera, P. J., Thangada, S., Tigyi, G., Osborne, D. A., Hla, T., Parrill, A. L., and Rosen, H. (2005) S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate, Chem Biol 12, 703-715.
7. Wei, S. H., Rosen, H., Matheu, M. P., Sanna, M. G., Wang, S. K., Jo, E., Wong, C. H., Parker, I., and Cahalan, M. D. (2005) Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses, Nat Immunol 6, 1228-1235.
8. Hla, T. (2003) Signaling and biological actions of sphingosine 1-phosphate, Pharmacol Res 47, 401-407.
9. Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J., Thornton, R., Shei, G. J., Card, D., Keohane, C., Rosenbach, M., Hale, J., Lynch, C. L., Rupprecht, K., Parsons, W., and Rosen, H. (2002) Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists, Science 296, 346-349.
10. Sanchez, T., and Hla, T. (2004) Structural and functional characteristics of S1P receptors, J Cell Biochem 92, 913-922.
11. Kon, J., Sato, K., Watanabe, T., Tomura, H., Kuwabara, A., Kimura, T., Tamama, K., Ishizuka, T., Murata, N., Kanda, T., Kobayashi, I., Ohta, H., Ui, M., and Okajima, F. (1999) Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells, J Biol Chem 274, 23940-23947.
12. Okamoto, H., Takuwa, N., Yatomi, Y., Gonda, K., Shigematsu, H., and Takuwa, Y. (1999) EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16, Biochem Biophys Res Commun 260, 203-208.
13. Windh, R. T., Lee, M. J., Hla, T., An, S., Barr, A. J., and Manning, D. R. (1999) Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins, J Biol Chem 274, 27351-27358.
14. Ishii, I., Friedman, B., Ye, X., Kawamura, S., McGiffert, C., Contos, J. J., Kingsbury, M. A., Zhang, G., Brown, J. H., and Chun, J. (2001) Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3, J Biol Chem 276, 33697-33704.
15. Zhang, G., Contos, J. J., Weiner, J. A., Fukushima, N., and Chun, J. (1999) Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate, Gene 227, 89-99.
16. Yamaguchi, F., Tokuda, M., Hatase, O., and Brenner, S. (1996) Molecular cloning of the novel human G protein-coupled receptor (GPCR) gene mapped on chromosome 9, Biochem Biophys Res Commun 227, 608-614.
17. Murakami, A., Takasugi, H., Ohnuma, S., Koide, Y., Sakurai, A., Takeda, S., Hasegawa, T., Sasamori, J., Konno, T., Hayashi, K., Watanabe, Y., Mori, K., Sato, Y., Takahashi, A., Mochizuki, N., and Takakura, N. (2010) Sphingosine 1-phosphate (S1P) regulates vascular contraction via S1P3 receptor: investigation based on a new S1P3 receptor antagonist, Mol Pharmacol 77, 704-713.
18. Niessen, F., Schaffner, F., Furlan-Freguia, C., Pawlinski, R., Bhattacharjee, G., Chun, J., Derian, C. K., Andrade-Gordon, P., Rosen, H., and Ruf, W. (2008) Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation, Nature 452, 654-658.


Sphingosine Receptor, Sphingosine-1-phosphate receptor 3, S1P3, endothelial differentiation sphingolipid G-protein-coupled receptor 3, EDG3, Sphingosine-1-phosphate receptor 2, S1P2, S1PR2, agonist, activator, GPCR, CHO, beta-lactamase, BLA, reporter gene, endothelial differentiation, 384, counterscreen, Tango, FRET, fluorescence, counterscreen, late stage, late stage AID, bradycardia, hypertension, powders, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLSCN
Assay Overview:

The purpose of this assay is to determine whether powder samples of compounds identified as active in the assay "Late-stage fluorescence-based cell-based dose response assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Synthesized compounds" (AID 540349) were nonselective agonists as assayed by activation of S1P2. A Chinese Hamster Ovary (CHO) cell line stably transfected with the human S1P2 receptor and a cAMP Response Element-beta lactamase (CRE-BLA) reporter construct was used to measure S1P2 agonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. Stimulation of the S1P2 receptor by agonist increases BLA gene transcription. This increase is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell-permeable BLA substrate. As designed, test compounds that act as S1P1 agonists will activate S1P1 and increase well FRET. Compounds were tested in quadruplicate at 50 uM.

Protocol Summary:

Cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media supplemented with 10% v/v heat inactivated dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 2 mg/mL Geneticin and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). Prior to assay, cells were suspended to a concentration of 1.25 million/mL in assay media, which consisted of phenol red-free Dulbecco's Modified Eagle's Media supplemented with 2% charcoal/dextran-treated fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin).

The assay was initiated by dispensing 10 uL of cell suspension to each test well of a 384 well plate (6,000 cells/well) followed by incubation at 37 C in 5% CO2 for 16 hrs. To the appropriate wells were then added 50 nL of test compound in DMSO (final nominal concentration of 50 uM, final DMSO concentration of 0.5%) or DMSO only (for high control wells) followed directly afterwards by 1 uL of S1P in 2% BSA (final concentration of 370 nM, i.e. a concentration that resulted in 80% activity). The high control (EC80 challenge) and low control (100% antagonism) were added to the appropriate control wells and plates were incubated again at 37 C in 5% CO2 for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer's protocol and 2.2 uL of this mixture was then added to each well. After a further 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and fluorescence emission wavelengths of 535 nm & 460 nm.

Prior to normalization, data were corrected by subtracting "background" for both emission channels (ie, fluorescence values from cell-free wells containing media and substrate only). To normalize assay data, these corrected values were used to calculate a ratio for each well, according to the following mathematical expression:

Ratio = I460 nm / I535 nm


I represents the measured fluorescence emission intensity at the enumerated wavelength.

The percent activation for each compound was calculated using well fluorescence as follows:

%_Activation = 100 * ( 1 - ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) ) )


Test_Compound is defined as wells containing test compound and S1P
Low_Control is defined as wells containing DMSO
High_Control is defined as wells containing 5uM S1P

In cases where treatment with 50 uM compound did not result in greater than 50% activation, the EC50 was determined manually as greater than 50 uM.

PubChem Activity Outcome and Score:

Compounds with an EC50 greater than 50 uM were considered inactive.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 0-0. There were no active compounds.

List of Reagents:

Dulbecco's Modified Eagle's Media with phenol red (Invitrogen, part 11965-092)
Dulbecco's Modified Eagle's Media without phenol red (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26400-044)
NEAA (Invitrogen, part 1114-050)
Sodium Pyruvate (Invitrogen, part 11360-070)
HEPES (Invitrogen, part 15630-080)
L-Glutamine (Invitrogen, part 25030-081)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055).
Charcoal/dextran treated fetal bovine serum (Hyclone, part SH30068.03)
Probenicid (Sigma, part P8761)
S1P agonist (Biomol, part SL-140)
LiveBLAzer (Invitrogen, part K1096)
JTE013 (Tocris, part 2392 ) 384-well plates (Greiner, part 788092)
T175 tissue culture flasks (Corning, part 431080)
In this assay, S1P had a 50% effective concentration (EC50) of approximately 50 nM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate beta-arrestin or BLA activity, and compounds that quench or emit fluorescence.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentrationString
2EC50*The concentration at which 50 percent of the activity in the antagonist assay is observed; (EC50) shown in micromolar.FloatμM
3Activation at 50 uM [1] (50μM**)Value of % Activation at 50 uM activator concentration [1]Float%
4Activation at 50 uM [2] (50μM**)Value of % Activation at 50 uM activator concentration [2]Float%
5Activation at 50 uM [3] (50μM**)Value of % Activation at 50 uM activator concentration [3]Float%
6Activation at 50 uM [4] (50μM**)Value of % Activation at 50 uM activator concentration [4]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: U01 AI074564

Data Table (Concise)