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
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.
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, 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
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.
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
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)
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
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: U-2 OS
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)