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BioAssay: AID 489009

Late-stage results from the probe development effort to identify antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): luminescence-based cell-based dose response assay to determine cytotoxicity of antagonist compounds

Name: Late-stage results from the probe development effort to identify antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): luminescence-based cell-based dose response assay to determine cytotoxicity of antagonist compounds. ..more
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 Related BioAssays
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AID: 489009
Data Source: The Scripps Research Institute Molecular Screening Center (U2O2CYTOX_INH_LUMI_384_6XCC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-11-17

Data Table ( Complete ):           View All Data
Tested Compound:
Related Experiments
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AIDNameTypeProbeComment
1510Primary Cell-Based Assay to Identify Antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Screening depositor-specified cross reference: Primary screen (S1P4 antagonists in singlicate)
1524Confirmation cell-based high throughput assay for antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Screening depositor-specified cross reference: Confirmation screen (S1P4 antagonists in triplicate)
1564Counterscreen assay for S1P4 antagonists: Cell-based high throughput screening assay to identify antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)Screening depositor-specified cross reference: Counterscreen (S1P1 antagonists in triplicate)
1692Fluorescence dose response cell-based high throughput screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Confirmatory depositor-specified cross reference: Dose response (S1P4 antagonists in triplicate)
1821Fluorescence-based counterscreen assay for S1P4 antagonists: Cell-based dose response high throughput screening assay to identify antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)Confirmatory depositor-specified cross reference: Dose response counterscreen (S1P1 antagonists in triplicate)
1853Summary of probe development efforts to identify antagonists of Sphingosine 1-Phosphate Receptor 4 (S1P4)Summary1 depositor-specified cross reference: Summary AID (S1P4 antagonists)
2332Late stage fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Confirmatory depositor-specified cross reference: Dose response (S1P4 antagonists in triplicate)
2346Late stage fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized analoguesConfirmatory depositor-specified cross reference: Late stage dose response (S1P4 antagonists in triplicate)
2349Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3)Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (S1P3 antagonists in triplicate)
2350Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 5 (S1P5)Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (S1P5 antagonists in triplicate)
2351Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (S1P1 antagonists in triplicate)
2354Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 2 (S1P2)Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (S1P2 antagonists in triplicate)
489017Counterscreen panel assay for S1P4 antagonists: Ricerca HitProfilingScreen + CYP450Other same project related to Summary assay
Description:
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 Fast Track
Grant Proposal PI: Michael Oldstone, TSRI
External Assay ID: U2O2CYTOX_INH_LUMI_384_6XCC50

Name: Late-stage results from the probe development effort to identify antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): luminescence-based cell-based dose response assay to determine cytotoxicity of antagonist compounds.

Description:

Pandemic influenza represents a significant public health threat, due in part to immune cell-mediated lung tissue damage induced during viral infection. Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets and serves to influence endothelial integrity, lung epithelial integrity (1), and lymphocyte recirculation (2-5) through five related high affinity G-protein coupled receptors. Recently, modulation of S1P receptors locally in the lungs was shown to alter dendritic cell activation and accumulation in the mediastinal lymph nodes, resulting in blunted T cell responses and control of immunopathological features of influenza virus infection (6). Reports showing that S1P5 expression is very low in dendritic cells but that S1P4 is highly expressed (7), suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gai and Gao G-proteins and activates ERK MAPK and PLC downstream pathways (8), indicating that selective antagonists of S1P4 may also serve as useful tools for understanding S1P4 biological function.

References:

1. Sanna, M.G., J. Liao, E. Jo, C. Alfonso, M.Y. Ahn, M.S. Peterson, B. Webb, S. Lefebvre, J. Chun, N. Gray, and H. Rosen, Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate. J Biol Chem, 2004. 279(14): p. 13839-48.
2. Forrest, M., S.Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, J. Hale, C. Keohane, C. Meyers, J. Milligan, S. Mills, N. Nomura, H. Rosen, M. Rosenbach, G.J. Shei, Singer, II, M. Tian, S. West, V. White, J. Xie, R.L. Proia, and S. Mandala, Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes. J Pharmacol Exp Ther, 2004. 309(2): p. 758-68.
3. Gon, Y., M.R. Wood, W.B. Kiosses, E. Jo, M.G. Sanna, J. Chun, and H. Rosen, S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF. Proc Natl Acad Sci U S A, 2005. 102(26): p. 9270-5.
4. Wei, S.H., H. Rosen, M.P. Matheu, M.G. Sanna, S.K. Wang, E. Jo, C.H. Wong, I. Parker, and M.D. Cahalan, Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat Immunol, 2005. 6(12): p. 1228-35.
5. Alfonso, C., M.G. McHeyzer-Williams, and H. Rosen, CD69 down-modulation and inhibition of thymic egress by short- and long-term selective chemical agonism of sphingosine 1-phosphate receptors. Eur J Immunol, 2006. 36(1): p. 149-59.
6. Jo, E., M.G. Sanna, P.J. Gonzalez-Cabrera, S. Thangada, G. Tigyi, D.A. Osborne, T. Hla, A.L. Parrill, and H. Rosen, S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. Chem Biol, 2005. 12(6): p. 703-15.
7. Maeda, Y., Matsuyuki, H., Shimano, K., Kataoka, H., Sugahara, K., and Chiba, K., Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3. J Immunol, 2007. 178(6): p. 3437-46.
8. Toman, R.E. and S. Spiegel, Lysophospholipid receptors in the nervous system. Neurochem Res, 2002. 27(7-8): p. 619-27.

Keywords:

Sphingosine Receptor, Sphingosine-1-phosphate receptor 4, S1P4, EDG6, LPC1, antagonist, inhibitor, GPCR, 384, luminescence, U2OS, cytotoxicity, CellTitre-Glo, CC50, late stage, late stage AID, powders, dose response, synthesized analogue, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to determine cytotoxicity of a powder sample of a compound identified being a potent and selective antagonist of S1P4. 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 six replicates 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 100 ug/ml (20 uL, 200,000 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 uM in growth media) were made. 5uL of diluted compound or media were added to wells, giving final compound concentrations of 0 - 20 uM. The plate was spun for 1 minute at 1000 rpm, then incubated at 37 C in a humidified incubator for 6 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 % growth inhibition for each well was then calculated as follows:
% Growth Inhibition = ( 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 growth inhibition 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% growth inhibition 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).
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
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:
U2OS 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 with a synthesized compound. 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 non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well.
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 micromolar.FloatμM
3Inhibition at 20 uM [1] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate one.Float%
4Inhibition at 20 uM [2] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate two.Float%
5Inhibition at 20 uM [3] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate three.Float%
6Inhibition at 20 uM [4] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate four.Float%
7Inhibition at 20 uM [5] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate five.Float%
8Inhibition at 20 uM [6] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate six.Float%
9Inhibition at 6.67 uM [1] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate one.Float%
10Inhibition at 6.67 uM [2] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate two.Float%
11Inhibition at 6.67 uM [3] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate three.Float%
12Inhibition at 6.67 uM [4] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate four.Float%
13Inhibition at 6.67 uM [5] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate five.Float%
14Inhibition at 6.67 uM [6] (6.67μM**)Value of % inhibition of growth at 6.67 uM inhibitor concentration; replicate six.Float%
15Inhibition at 2.22 uM [1] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate one.Float%
16Inhibition at 2.22 uM [2] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate two.Float%
17Inhibition at 2.22 uM [3] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate three.Float%
18Inhibition at 2.22 uM [4] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate four.Float%
19Inhibition at 2.22 uM [5] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate five.Float%
20Inhibition at 2.22 uM [6] (2.22μM**)Value of % inhibition of growth at 2.22 uM inhibitor concentration; replicate six.Float%
21Inhibition at 0.741 uM [1] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate one.Float%
22Inhibition at 0.741 uM [2] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate two.Float%
23Inhibition at 0.741 uM [3] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate three.Float%
24Inhibition at 0.741 uM [4] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate four.Float%
25Inhibition at 0.741 uM [5] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate five.Float%
26Inhibition at 0.741 uM [6] (0.741μM**)Value of % inhibition of growth at 0.741 uM inhibitor concentration; replicate six.Float%
27Inhibition at 2.47 uM [1] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate one.Float%
28Inhibition at 2.47 uM [2] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate two.Float%
29Inhibition at 2.47 uM [3] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate three.Float%
30Inhibition at 2.47 uM [4] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate four.Float%
31Inhibition at 2.47 uM [5] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate five.Float%
32Inhibition at 2.47 uM [6] (0.247μM**)Value of % inhibition of growth at 0.247 uM inhibitor concentration; replicate six.Float%
33Inhibition at 0.0823 uM [1] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate one.Float%
34Inhibition at 0.0823 uM [2] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate two.Float%
35Inhibition at 0.0823 uM [3] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate three.Float%
36Inhibition at 0.0823 uM [4] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate four.Float%
37Inhibition at 0.0823 uM [5] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate five.Float%
38Inhibition at 0.0823 uM [6] (0.0823μM**)Value of % inhibition of growth at 0.0823 uM inhibitor concentration; replicate six.Float%
39Inhibition at 0.0274 uM [1] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate one.Float%
40Inhibition at 0.0274 uM [2] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate two.Float%
41Inhibition at 0.0274 uM [3] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate three.Float%
42Inhibition at 0.0274 uM [4] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate four.Float%
43Inhibition at 0.0274 uM [5] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate five.Float%
44Inhibition at 0.0274 uM [6] (0.0274μM**)Value of % inhibition of growth at 0.0274 uM inhibitor concentration; replicate six.Float%
45Inhibition at 0 uM [1] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate one.Float%
46Inhibition at 0 uM [2] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate two.Float%
47Inhibition at 0 uM [3] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate three.Float%
48Inhibition at 0 uM [4] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate four.Float%
49Inhibition at 0 uM [5] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate five.Float%
50Inhibition at 0 uM [6] (0μM**)Value of % inhibition of growth at 0 uM inhibitor concentration; replicate six.Float%

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

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