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BioAssay: AID 463225

Late-stage fluorescence dose response cell-based counterscreening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist

Name: Late-stage fluorescence dose response cell-based counterscreening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist. ..more
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 Tested Compounds
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Active(1)
 
 
AID: 463225
Data Source: The Scripps Research Institute Molecular Screening Center (S1P4_AG_BLA_384_3XIC50_Antagonist)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-16
Hold-until Date: 2011-04-12
Modify Date: 2011-10-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
1509Primary Cell-Based Assay to Identify Agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Screening depositor-specified cross reference: Primary screen (S1P4 agonists in singlicate)
1523Confirmation cell-based high throughput assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Screening depositor-specified cross reference: Confirmation screen (S1P4 agonists in triplicate)
1563Counterscreen assay for S1P4 agonists: Cell-based high throughput screening assay to identify agonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)Screening depositor-specified cross reference: Counterscreen (S1P1 agonists in triplicate)
1686Fluorescence dose response cell-based high throughput screening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)Confirmatory depositor-specified cross reference: Dose response screen (S1P4 agonists in triplicate)
1701Fluorescence-based counterscreen assay for S1P4 agonists: Cell-based dose response high throughput screening assay to identify agonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)Confirmatory depositor-specified cross reference: Counterscreen dose response (S1P1 agonists in triplicate)
1801Summary of probe development efforts to identify agonists of Sphingosine 1-Phosphate Receptor 4 (S1P4)Summary2 depositor-specified cross reference: Summary AID (S1P4 agonists)
463107Late-stage fluorescence dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compoundsConfirmatory depositor-specified cross reference: Late-stage screen dose response (S1P4 agonsts in triplicate)
463118Late-stage fluorescence-based dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 1 (S1P1) counterscreen assayConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (S1P1 agonists in triplicate)
463119Late-stage assay provider results from the probe development effort to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): luminescence-based cell-based dose response assay to determine cytotoxicity of agonist compoundsConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (Cytotoxicity in quadruplicate)
463122Late-stage fluorescence-based dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 2 (S1P2) counterscreen assayConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (S1P2 agonists in triplicate)
463123Late-stage fluorescence-based dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 3 (S1P3) counterscreen assayConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (S1P3 agonists in triplicate)
463129Late-stage fluorescence-based dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 5 (S1P5) counterscreen assayConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (S1P5 agonists in triplicate)
504400Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450Other depositor-specified cross reference
504867Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 5 (S1P5) agonist assay Set 2Confirmatory depositor-specified cross reference
504869Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 5 (S1P5) antagonist assay Set 2Confirmatory depositor-specified cross reference
504870Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 1 (S1P1) agonist assay Set 2Confirmatory depositor-specified cross reference
504871Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist controlConfirmatory depositor-specified cross reference
504872Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 3 (S1P3) antagonist assay Set 2Confirmatory depositor-specified cross reference
504873Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 2 (S1P2) agonist assay Set 2Confirmatory depositor-specified cross reference
504875Late-stage assay provider results from the probe development effort to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): luminescence-based cell-based dose response assay to determine cytotoxicity of agonist compounds Set 2Confirmatory depositor-specified cross reference
504876Late-stage fluorescence dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 4 (S1P4) antagonist assay Set 2Confirmatory depositor-specified cross reference
504877Late-stage fluorescence dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compounds set 2Other depositor-specified cross reference
504879Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 3 (S1P3) agonist assay Set 2Confirmatory depositor-specified cross reference
504880Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 1 (S1P1) antagonist assayConfirmatory depositor-specified cross reference
504881Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Sphingosine 1-Phosphate Receptor 2 (S1P2) antagonist assay Set 2Confirmatory depositor-specified cross reference
504917Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 1.2 nMConfirmatory depositor-specified cross reference
504918Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 0.4 nMConfirmatory depositor-specified cross reference
504919Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 11 nMConfirmatory depositor-specified cross reference
504921Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 33 nMConfirmatory depositor-specified cross reference
504923Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 100 nMConfirmatory depositor-specified cross reference
504924Late-stage fluorescence-based dose-response cell-based counterscreen assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist 3.7 nMConfirmatory depositor-specified cross reference
540332Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450: Set 2Other depositor-specified cross reference
504460Late-stage fluorescence dose response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): purchased compounds EC50Confirmatory 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: S1P4_AG_BLA_384_3XIC50_Antagonist

Name: Late-stage fluorescence dose response cell-based counterscreening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonist.

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, agonist, activator, GPCR, 384, antagonist, Tango, FRET, GAL4-VP16, beta-arrestin, beta-lactamase, BLA, reporter gene, fluorescence, dose response, late stage, late stage AID, powders, 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 whether a test compound with S1P4 agonist activity is inhibited by an S1P4-selective antagonist. This assay uses Tango S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) 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 S1P4 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. The presence of an S1P4-selective antagonist compound in the assay inhibits S1P4 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. In the presence of a test agonist compound, the S1P4 receptor will be stimulated, leading to an increase in well FRET. Compound was tested in triplicate using a 7-point, 1:3 dilution series starting at a nominal concentration of 10 uM.
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 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 uL of cell suspension to each well, followed by overnight incubation at 37 C in 5% CO2 and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, 370 nM S1P4-selective antagonist SR2-304 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 inhibition for each compound was calculated using well fluorescence as follows:
% Inhibition = 100 * ( 1 - ( ( Median_Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) )
Where:
Test_Compound is defined as wells containing test compound and SR2-304.
Low_Control is defined as wells containing SR2-304.
High_Control is defined as wells containing DMSO only.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 10 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 10 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent inhibition value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent inhibition value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
Tango EDG6-bla U2OS cells (Invitrogen, part K1622)
S1P4-selective antagonist SR2-304 (provided by SRIMSC)
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)
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)
Comment
All data reported were normalized on a per-plate basis. 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
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
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
2Inhibition at 10 uM [1] (10μM**)Value of % Inhibition at 10 uM S1P4-selective antagonist concentration; replicate one.Float%
3Inhibition at 10 uM [2] (10μM**)Value of % Inhibition at 10 uM S1P4-selective antagonist concentration; replicate two.Float%
4Inhibition at 10 uM [3] (10μM**)Value of % Inhibition at 10 uM S1P4-selective antagonist concentration; replicate three.Float%
5Inhibition at 3.3 uM [1] (3.3μM**)Value of % Inhibition at 3.3 uM S1P4-selective antagonist concentration; replicate one.Float%
6Inhibition at 3.3 uM [2] (3.3μM**)Value of % Inhibition at 3.3 uM S1P4-selective antagonist concentration; replicate two.Float%
7Inhibition at 3.3 uM [3] (3.3μM**)Value of % Inhibition at 3.3 uM S1P4-selective antagonist concentration; replicate three.Float%
8Inhibition at 1.1 uM [1] (1.1μM**)Value of % Inhibition at 1.1 uM S1P4-selective antagonist concentration; replicate one.Float%
9Inhibition at 1.1 uM [2] (1.1μM**)Value of % Inhibition at 1.1 uM S1P4-selective antagonist concentration; replicate two.Float%
10Inhibition at 1.1 uM [3] (1.1μM**)Value of % Inhibition at 1.1 uM S1P4-selective antagonist concentration; replicate three.Float%
11Inhibition at 0.370 uM [1] (0.37μM**)Value of % Inhibition at 0.370 uM S1P4-selective antagonist concentration; replicate one.Float%
12Inhibition at 0.370 uM [2] (0.37μM**)Value of % Inhibition at 0.370 uM S1P4-selective antagonist concentration; replicate two.Float%
13Inhibition at 0.370 uM [3] (0.37μM**)Value of % Inhibition at 0.370 uM S1P4-selective antagonist concentration; replicate three.Float%
14Inhibition at 0.124 uM [1] (0.124μM**)Value of % Inhibition at 0.124 uM S1P4-selective antagonist concentration; replicate one.Float%
15Inhibition at 0.124 uM [2] (0.124μM**)Value of % Inhibition at 0.124 uM S1P4-selective antagonist concentration; replicate two.Float%
16Inhibition at 0.124 uM [3] (0.124μM**)Value of % Inhibition at 0.124 uM S1P4-selective antagonist concentration; replicate three.Float%
17Inhibition at 0.041 uM [1] (0.041μM**)Value of % Inhibition at 0.041 uM S1P4-selective antagonist concentration; replicate one.Float%
18Inhibition at 0.041 uM [2] (0.041μM**)Value of % Inhibition at 0.041 uM S1P4-selective antagonist concentration; replicate two.Float%
19Inhibition at 0.041 uM [3] (0.041μM**)Value of % Inhibition at 0.041 uM S1P4-selective antagonist concentration; replicate three.Float%
20Inhibition at 0 uM [1] (0μM**)Value of % Inhibition with no S1P4-selective antagonist present; replicate one.Float%
21Inhibition at 0 uM [2] (0μM**)Value of % Inhibition with no S1P4-selective antagonist present; replicate two.Float%
22Inhibition at 0 uM [3] (0μM**)Value of % Inhibition with no S1P4-selective antagonist present; replicate three.Float%

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

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