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
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: S1P1_AG_BLA_384_3XEC50_SET 3
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 1 (S1P1) agonist assay Set 3.
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 1, S1P1, EDG1, agonist, activator, GPCR, 384, Tango, FRET, GAL4-VP16, beta-arrestin, beta-lactamase, BLA, reporter gene, fluorescence, primary, 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 dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compounds" (AID 540349) were nonselective agonists as assayed by activation of S1P1. This assay uses Tango S1P1-bla U2OS cells which express the human Endothelial Differentiation Gene 1 (EDG1; S1P1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression 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 triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 uM in Experiment 1, and 50 uM in Experiments 2 and 3. Protocol Summary: U2OS cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 ug/mL Zeocin, 50 ug/mL Hygromycin, and 100 ug/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 uL of cell suspension to each well of a 384 well plate (10,000 cells/well), followed by overnight incubation at 37 C in 5% CO2 and 95% RH. The next day, 50 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (40 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 C in 5% CO2 for 4 hours. After the incubation, 2.2 uL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 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 Where: 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 ) ) ) Where: 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 5 uM S1P Percent activation was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated EC50 values were reported. In cases where the highest concentration tested (i.e. 10 uM in Experiment 1 and 50 uM in Experiments 2 and 2) did not result in greater than 50% activation, the EC50 was determined manually as greater than the highest concentration tested. In cases where the software was not able to generate a curve, "Not Converged" is reported for the statistical values. PubChem Activity Outcome and Score: The following applies to each panel in this assay: Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores. Experiment 1 Score: The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0. Experiment 2 Score: The PubChem Activity Score range for active compounds is 100-84, and for inactive compounds 41-0. Experiment 3 Score: The PubChem Activity Score range for active compounds is 100-87, and for inactive compounds 82-0. Overall Outcome and Score: Compounds that were active in all experiments were considered active, otherwise they were considered inactive. The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100. The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds. The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0. List of Reagents: Tango EDG1-bla U2OS cells (Invitrogen, part K1520) GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (Invitrogen, part K1025) Probenecid (Sigma, part P8761) Freestyle Expression Medium (Assay media; Invitrogen, part 12338-018) McCoy's 5A Medium (modified) (1X) (Invitrogen, 16600-082) Fetal Bovine Serum, dialyzed (Invitrogen, part 26400-036) NEAA (Invitrogen, part 1114-050) Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15140-122) Sodium Pyruvate (Invitrogen, part 11360-070) PBS without calcium or magnesium (Invitrogen, part 14190-136) HEPES (Invitrogen, part 15630-080) Trypsin/EDTA (Invitrogen, part 25300-054) S1P (Avanti Polar Lipids, part 860492P) Fatty Acid Free BSA (Calbiochem, part NC9734015) Zeocin (Invitrogen, part R250-01) Hygromycin (Invitrogen, part 10687-010) Geneticin (Invitrogen, part 10131-027) 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.
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