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BioAssay: AID 1701

Fluorescence-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)

Name: Fluorescence-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) ..more
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 Tested Compounds
 Tested Compounds
All(46)
 
 
Active(15)
 
 
Inactive(31)
 
 
 Tested Substances
 Tested Substances
All(46)
 
 
Active(15)
 
 
Inactive(31)
 
 
AID: 1701
Data Source: The Scripps Research Institute Molecular Screening Center (S1P1_AG_BLA_1536_3XEC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-29
Modify Date: 2010-11-10

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 15
Depositor Specified Assays
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AIDNameTypeProbeComment
1509Primary Cell-Based Assay to Identify Agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)screening Primary screen
1523Confirmation cell-based high throughput assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)screening Confirmation assay
1801Summary of probe development efforts to identify agonists of Sphingosine 1-Phosphate Receptor 4 (S1P4)summary2
463107Late-stage fluorescence dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compoundsconfirmatory
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
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
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
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
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
463225Late-stage fluorescence dose response cell-based counterscreening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonistconfirmatory
504400Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450other
504460Late-stage fluorescence dose response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): purchased compounds EC50confirmatory
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
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
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
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
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
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
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
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
504877Late-stage fluorescence dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compounds set 2other
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
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
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
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
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
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
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
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
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
540332Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450: Set 2other
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Oldstone, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: U01 AI074564 Fast Track
Grant Proposal PI: Michael Oldstone, TSRI

External Assay ID: S1P1_AG_BLA_1536_3XEC50

Name: Fluorescence-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)

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 Galphai and Galphao G-proteins and activates ERK MAPK and PLC downstream pathways (7). Thus, the identification of compounds that act as selective S1P4 agonists will provide insight into S1P4 biology and may serve as useful tools to limit lung tissue injury resulting from influenza infection.

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, S1PR4, EDG6, LPC1, agonist, activator, HTS, GPCR, 1536, Tango, FRET, GAL4-VP16, beta-arrestin, beta-lactamase, BLA, reporter gene, fluorescence, dose response, counterscreen, S1P1, EDG1, EDG-1, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Probe Production Center Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary Cell-Based Assay to Identify Agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)" (PubChem AID 1509), and that confirmed activity in a set of experiments entitled, "Confirmation Cell-Based Assay to Identify Agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)" (PubChem AID 1523). This assay also serves as a counterscreen to determine whether these compounds are nonselective agonists due to activation of S1P1. This assay uses Tango S1P1-bla U2OS cells which express S1P1 (EDG1) 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 50 uM.

Protocol Summary:

U2OS cells were cultured in T-175 sq cm flasks at 37 degrees 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 micrograms/mL Zeocin, 50 micrograms/mL Hygromycin, 100 micrograms/mL Geneticin.

Prior to the start of the assay, cells were suspended at a concentration of 625,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 ul of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO2 and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (5 uM final concentration) prepared in 2% fatty-acid free BSA, was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO2 for 4 hours. After the incubation, 1 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* ((Test_Compound - Median_Low_Control)/ (Median_High_Control - Median_Low_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 S1P.

For each test compound, percent activation 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 EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 50 micromolar) did not result in greater than 50% activation, the EC50 was determined manually depending on the observed activation at the individual concentrations. Compounds with EC50 values greater than 10 uM were considered inactive. Compounds with EC50 values equal to or less than 10 uM were considered active.

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, with the most potent compounds assigned the highest activity scores.

The inactive compounds of this assay have activity score range of 0 to 51 and active compounds range of activity score is 54 to 100.

List of Reagents:

Tango EDG-1-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)
1536-well plates (Greiner, part 789072)
T175 tissue culture flasks (Corning, part 431080)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, S1P had a 50% effective concentration (EC50) of approximately 5 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. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentrationString
2EC50*The concentration at which 50 percent of the activity in the agonist assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) from the agonist assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is Float steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent Activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Activation at 0.003 uM (0.003μM**)Value of %Activation at 0.003 uM activator concentration; average of triplicate measurement.Float%
12Activation at 0.008 nM (0.008μM**)Value of %Activation at 0.008 uM activator concentration; average of triplicate measurement.Float%
13Activation at 0.02 uM (0.02μM**)Value of %Activation at 0.02 uM activator concentration; average of triplicate measurement.Float%
14Activation at 0.07 uM (0.07μM**)Value of %Activation at 0.07 uM activator concentration; average of triplicate measurement.Float%
15Activation at 0.2 uM (0.2μM**)Value of %Activation at 0.2 uM activator concentration; average of triplicate measurement.Float%
16Activation at 0.6 uM (0.6μM**)Value of %Activation at 0.6 uM activator concentration; average of triplicate measurement.Float%
17Activation at 2.0 uM (2μM**)Value of %Activation at 2 uM activator concentration; average of triplicate measurement.Float%
18Activation at 6.0 uM (6μM**)Value of %Activation at 6 uM activator concentration; average of triplicate measurement.Float%
19Activation at 20.0 uM (30μM**)Value of %Activation at 20 uM activator concentration; average of triplicate measurement.Float%
20Activation at 50.0 uM (50μM**)Value of %Activation at 50 uM activator concentration; average of triplicate measurement.Float%

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

Data Table (Concise)
Classification
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