Late-stage assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): radiometric-based cell-based dose response S1P agonist competition binding assay
Name: Late-stage assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): radiometric-based cell-based dose response S1P agonist competition binding assay. ..more
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Oldstone, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: U01 AI074564
Grant Proposal PI: Michael Oldstone, TSRI
External Assay ID: S1P3_AG_BLA_384_3XIC50
Name: Late-stage assay to identify agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): radiometric-based cell-based dose response S1P agonist competition binding 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, agonist, activator, GPCR, CHO cells, competition binding assay, binding assay, 24, radiometric assay, late stage, late stage AID, powders, bradycardia, hypertension, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLSCN
§ Panel component ID.
The purpose of this assay is to determine whether a powder compound with S1P3 agonist activity competes competitively or noncompetitively for binding of [33P]S1P to cells that contain the S1P3 receptor. In this assay, S1P3 Jump-In Chinese Hamster Ovary (CHO) cells containing human S1P3 are incubated with a fixed concentration of [33P]S1P and increasing concentrations of S1P3 agonist. If the S1P3 agonist compound competes with the [33P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will decrease as the concentration of cold S1P3 increases. If the S1P3 agonist compound does not compete with the [33P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will remain unchanged as the concentration of S1P3 agonist increases. S1P3 agonist compound was tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 uM (S1P3 Experiment). An experiment to demonstrate that cold S1P competes competitively for binding of [33P]S1P to cells that contain the S1P3 receptor serves as a control (Control Experiment). In this assay, S1P3 Jump-In Chinese Hamster Ovary (CHO) cells containing human S1P3 are incubated with a fixed concentration of [33P]S1P and increasing concentrations of cold S1P. If the cold S1P competes with the [33P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will decrease as the concentration of cold S1P3 increases. Cold S1P was tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 uM.
S1P3 Jump-In CHO 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 (DMEM) (with GlutaMAX) containing 10% v/v heat inactivated fetal bovine serum (dialyzed), 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, and 1x penicillin-streptomycin-neomycin. On the day before the assay, cells were suspended at a concentration of 0.2 x 10^6/mL in the growth media and plated at 0.1 x 10^6/well in a 24-well plate. On the day of the assay, the growth medium was replaced with serum-starvation medium consisted of Dulbecco's Modified Eagle's Media (DMEM) (with GlutaMAX) containing 0.1 mM NEAA, 1 mM sodium pyruvate, and no antibiotics. The cells were incubated at 37 C for 4 hours.
After 4 hours of serum starvation, the medium was removed and the cells were rinsed with 200 uL of ice-cold binding buffer. The binding buffer consisted of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na3VO4, 0.5% fatty acid-free BSA, and 1x protease inhibitor cocktail.
S1P3 Experiment: For the S1P3 agonist competition assay, 30 uL of increasing concentrations of SID 124360653 (final SID 124360653 concentrations of 0.001 nM to 10 uM) and 270 uL ice-cold binding buffer containing [33P]S1P (final concentration of 0.1 nM) were added to the cells in each well.
Control Experiment: For the control assay, 30 uL of increasing concentrations of cold S1P (final S1P concentrations of 0.001 nM to 10 uM) and 270 uL ice-cold binding buffer containing [33P]S1P (final concentration of 0.1 nM) were added to the cells in each well.
Next, for both Experiments, the cells were incubated at 4 C for 30 minutes. The cells were then washed three times with 500 uL ice-cold binding buffer. The cells were lysed with 300 uL 0.5% SDS and transferred to scintillation vials. 5 mL of scintillation cocktail was dispensed into each vial and the vial vortexed. The 33P radioactivity (cpm) was counted for 5 minutes/vial in a Beckman LS 6000SC counter.
Prior to normalization, data were corrected by subtracting "background" counts (ie, background radioactivity from cells without 33P-S1P addition). To normalize assay data, 0% was defined as the smallest value in the data set and 100% as the largest value in the data set.
S1P3 Experiment: Percent binding was plotted against the log of the SID 124360653 concentration. A three parameter equation describing a nonlinear regression curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated IC50 values were reported.
Control Experiment: Percent binding was plotted against the log of the cold S1P concentration. A three parameter equation describing a nonlinear regression curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated IC50 values were reported.
PubChem Activity Outcome and Score:
S1P3 Experiment: Compounds with an IC50 greater than 10 uM were considered inactive for competitive inhibition, and the binding mode was considered to be noncompetitive. Compounds with an IC50 equal to or less than 10 uM were considered active for competitive inhibition, and the binding mode was considered to be competitive.
Control Experiment: Control experiment, no compound was tested.
S1P3 Experiment Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
Overall Outcome and Score:
If compounds were active in S1P3 Experiment, then they were considered active overall.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Dulbecco's Modified Eagle's Media + GlutaMAX(Invitrogen, part 10569-010)
Dialyzed Fetal Bovine Serum (Invitrogen, part 26400-044)
NEAA (Invitrogen, part 1114-050)
HEPES (Invitrogen, part 15630-080)
Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15140-055)
S1P (Biomol, part SL140-0001)
[33P]S1P (American Radiolabeled Chemicals, part ARP 0144)
Fatty Acid Free BSA (JHR, part 85041)
Complete, EDTA-free, Protease Inhibitor Cocktail Tablets (Roche, part 11 873 580 001)
24-well plates (Corning, part 3524)
T175 tissue culture flasks (Corning, part 431080)
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration. § Panel component ID.