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BioAssay: AID 540344

Late-stage assay provider results from the probe development effort to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of agonist compounds

Name: Late-stage assay provider results from the probe development effort to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of agonist compounds. ..more
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 Tested Compounds
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Inactive(1)
 
 
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Inactive(1)
 
 
 Related BioAssays
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AID: 540344
Data Source: The Scripps Research Institute Molecular Screening Center (U-2OSCYTOX_INH_LUMI_384_CC50_SET 3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-08-12
Hold-until Date: 2012-03-01
Modify Date: 2012-03-01

Data Table ( Complete ):           View All Data
Tested Compound:
Related Experiments
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AIDNameTypeProbeComment
373S1P3 Agonist Primary HTS and Confirmation AssaysScreening depositor-specified cross reference: Primary screen (S1P3 agonists in singlicate)
439S1P3 Agonist Dose-Response Potency AssayConfirmatory depositor-specified cross reference: Dose response (S1P3 agonists in triplicate)
1192Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Purchased AnaloguesConfirmatory depositor-specified cross reference: Dose response (S1P3 agonists in triplicate)
540309Summary of probe development efforts to identify agonists of Sphingosine 1-Phosphate Receptor 3 (S1P3)Summary5 depositor-specified cross reference: Summary (S1P3 agonists)
588327Late-stage assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): radiometric-based cell-based dose response S1P agonist competition binding assayConfirmatory depositor-specified cross reference
540349Late-stage fluorescence-based cell-based dose response assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Synthesized compoundsConfirmatory same project related to Summary assay
540351Late-stage counterscreen panel assay for S1P3 agonists: Ricerca HitProfilingScreen + CYP450Other same project related to Summary assay
540366Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 4 (S1P4) agonist assayConfirmatory same project related to Summary assay
540367Late-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 assayConfirmatory same project related to Summary assay
540368Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 1 (S1P1) agonist assay Set 3Confirmatory same project related to Summary assay
540369Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Sphingosine 1-Phosphate Receptor 5 (S1P5) agonist assayConfirmatory same project related to Summary assay
Description:
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: U-2OSCYTOX_INH_LUMI_384_CC50_SET 3

Name: Late-stage assay provider results from the probe development effort to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of agonist compounds.

Description:

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.

References:

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.

Keywords:

Sphingosine Receptor, Sphingosine-1-phosphate receptor 3, S1P3, endothelial differentiation sphingolipid G-protein-coupled receptor 3, EDG3, agonist, activator, luminescence, U2OS, cytotoxicity, CellTitre-Glo, CC50, late stage, late stage AID, powders, bradycardia, hypertension, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLSCN
Protocol
Assay Overview:

The purpose of this assay is to determine cytotoxicity of powder compounds identified as S1P3 agonists. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 uM.

Protocol Summary:

This assay was started by dispensing U2OS cells in McCoy's 5A medium plus 10% FBS, penicillin 100U/mL and streptomycin 100ug/mL (20 uL, 4 x 10E3 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 uM in growth media) were made. 5 uL of diluted compound or media were added to wells, giving final compound concentrations of 0 - 20 uM. The plate was incubated at 37 C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 uL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader.

The % Cell Viability for each well was then calculated as follows:

%_Cell_Viability = 1 - ( MedianRFU_High_Control - RFU_Test_Compound ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).

Percent Cell Viability was plotted against the log of the compound concentration. The CC50 is reported as ">X uM" (where X = the highest concentration tested for which >50% Cell Viability was observed).

PubChem Activity Outcome and Score:

Compounds with a CC50 value of less than 10 uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 uM were considered inactive (non-cytotoxic).

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 are no active compounds.

List of Reagents:

U-2OS cells (ATCC, part HTB-96)
McCoy's 5A Medium (Invitrogen, part 16600-082)
FBS (Invitrogen, part 26140-079)
Penicillin / Streptomycin (Invitrogen, part 15140-122)
Cell Titer-Glo (Promega, part G7572)
384-well plates (Corning 3570)
Comment
This assay was performed by the assay provider with powder samples of compounds. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: U-2 OS
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.String
2CC50*The value for concentration at which 50% of surviving cells are observed; CC50 shown in uM.FloatμM
3Cell Viability at 20 uM [1] (20μM**)Value of % Cell Viability at 20 uM compound concentration [1]Float%
4Cell Viability at 20 uM [2] (20μM**)Value of % Cell Viability at 20 uM compound concentration [2]Float%
5Cell Viability at 20 uM [3] (20μM**)Value of % Cell Viability at 20 uM compound concentration [3]Float%
6Cell Viability at 20 uM [4] (20μM**)Value of % Cell Viability at 20 uM compound concentration [4]Float%
7Cell Viability at 20 uM [5] (20μM**)Value of % Cell Viability at 20 uM compound concentration [5]Float%
8Cell Viability at 20 uM [6] (20μM**)Value of % Cell Viability at 20 uM compound concentration [6]Float%
9Cell Viability at 6.67 uM [1] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [1]Float%
10Cell Viability at 6.67 uM [2] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [2]Float%
11Cell Viability at 6.67 uM [3] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [3]Float%
12Cell Viability at 6.67 uM [4] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [4]Float%
13Cell Viability at 6.67 uM [5] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [5]Float%
14Cell Viability at 6.67 uM [6] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [6]Float%
15Cell Viability at 2.22 uM [1] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [1]Float%
16Cell Viability at 2.22 uM [2] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [2]Float%
17Cell Viability at 2.22 uM [3] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [3]Float%
18Cell Viability at 2.22 uM [4] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [4]Float%
19Cell Viability at 2.22 uM [5] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [5]Float%
20Cell Viability at 2.22 uM [6] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [6]Float%
21Cell Viability at 0.74 uM [1] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [1]Float%
22Cell Viability at 0.74 uM [2] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [2]Float%
23Cell Viability at 0.74 uM [3] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [3]Float%
24Cell Viability at 0.74 uM [4] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [4]Float%
25Cell Viability at 0.74 uM [5] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [5]Float%
26Cell Viability at 0.74 uM [6] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [6]Float%
27Cell Viability at 0.25 uM [1] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [1]Float%
28Cell Viability at 0.25 uM [2] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [2]Float%
29Cell Viability at 0.25 uM [3] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [3]Float%
30Cell Viability at 0.25 uM [4] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [4]Float%
31Cell Viability at 0.25 uM [5] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [5]Float%
32Cell Viability at 0.25 uM [6] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [6]Float%
33Cell Viability at 0.082 uM [1] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [1]Float%
34Cell Viability at 0.082 uM [2] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [2]Float%
35Cell Viability at 0.082 uM [3] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [3]Float%
36Cell Viability at 0.082 uM [4] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [4]Float%
37Cell Viability at 0.082 uM [5] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [5]Float%
38Cell Viability at 0.082 uM [6] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [6]Float%
39Cell Viability at 0.027 uM [1] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [1]Float%
40Cell Viability at 0.027 uM [2] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [2]Float%
41Cell Viability at 0.027 uM [3] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [3]Float%
42Cell Viability at 0.027 uM [4] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [4]Float%
43Cell Viability at 0.027 uM [5] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [5]Float%
44Cell Viability at 0.027 uM [6] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [6]Float%
45Cell Viability at 0 uM [1] (0μM**)Value of % Cell Viability at 0 uM compound concentration [1]Float%
46Cell Viability at 0 uM [2] (0μM**)Value of % Cell Viability at 0 uM compound concentration [2]Float%
47Cell Viability at 0 uM [3] (0μM**)Value of % Cell Viability at 0 uM compound concentration [3]Float%
48Cell Viability at 0 uM [4] (0μM**)Value of % Cell Viability at 0 uM compound concentration [4]Float%
49Cell Viability at 0 uM [5] (0μM**)Value of % Cell Viability at 0 uM compound concentration [5]Float%
50Cell Viability at 0 uM [6] (0μM**)Value of % Cell Viability at 0 uM compound concentration [6]Float%

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

Data Table (Concise)
Data Table ( Complete ):     View All Data
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