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BioAssay: AID 504870

Late-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 2

Name: Late-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 2. ..more
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 Tested Compounds
 Tested Compounds
All(12)
 
 
Active(10)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(12)
 
 
Active(10)
 
 
Inactive(2)
 
 
AID: 504870
Data Source: The Scripps Research Institute Molecular Screening Center (S1P1_AG_BLA_384_3XEC50_SET 2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-06-29
Hold-until Date: 2012-03-01
Modify Date: 2012-03-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 10
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 (S1P4 agonists in singlicate)
1523Confirmation cell-based high throughput assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)screening 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 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 Dose response (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 Dose response counterscreen (S1P1 agonists in triplicate)
1801Summary of probe development efforts to identify agonists of Sphingosine 1-Phosphate Receptor 4 (S1P4)summary2 Summary (S1P4 agonists)
463107Late-stage fluorescence dose-response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): Synthesized compoundsconfirmatory Late stage dose response (S1P4 agonists 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 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 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 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 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 Late stage dose response counterscreen (S1P5 agonists in triplicate)
463225Late-stage fluorescence dose response cell-based counterscreening assay for agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): inhibition by S1P4-selective antagonistconfirmatory Late stage dose response counterscreen (inhibition by S1P4-selective antagonist)
504400Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450other Late-stage counterscreen panel assay (Ricerca hit profiling CYP450)
504460Late-stage fluorescence dose response cell-based assay to identify agonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4): purchased compounds EC50confirmatory Late-stage dose response (S1P4 agonists in triplicate)
540332Late-stage counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450: Set 2other
Description:
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 2

Name: Late-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 2.

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). 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, Sphingosine-1-phosphate receptor 1, S1P1, EDG6, EDG1, LPC1, agonist, activator, GPCR, 384, Tango, FRET, GAL4-VP16, beta-arrestin, beta-lactamase, BLA, reporter gene, fluorescence, primary, late stage, late stage AID, powders, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLSCN
Protocol
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 463107) 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 25 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 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. 25 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 25 uM.

PubChem Activity Outcome and Score:

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.

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 active compounds is 100-1 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)
Comment
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.
Categorized Comment
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: = 100 / ( 1 + 10^( ( [LogEC50] - Log( [Concentration] * 10^-6 ) * [Hill Slope] ) )

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 antagonist assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Value of Log EC50Float
4Hill SlopeValue of Hill SlopeFloat
5R squaredValue of R squaredFloat
6Activation at 25 uM [1] (25μM**)Value of % activation at 25 uM; replicate [1]Float%
7Activation at 25 uM [2] (25μM**)Value of % activation at 25 uM; replicate [2]Float%
8Activation at 25 uM [3] (25μM**)Value of % activation at 25 uM; replicate [3]Float%
9Activation at 8.3 uM [1] (8.3μM**)Value of % activation at 8.3 uM; replicate [1]Float%
10Activation at 8.3 uM [2] (8.3μM**)Value of % activation at 8.3 uM; replicate [2]Float%
11Activation at 8.3 uM [3] (8.3μM**)Value of % activation at 8.3 uM; replicate [3]Float%
12Activation at 2.8 uM [1] (2.8μM**)Value of % activation at 2.8 uM; replicate [1]Float%
13Activation at 2.8 uM [2] (2.8μM**)Value of % activation at 2.8 uM; replicate [2]Float%
14Activation at 2.8 uM [3] (2.8μM**)Value of % activation at 2.8 uM; replicate [3]Float%
15Activation at 0.933 uM [1] (0.933μM**)Value of % activation at 0.933 uM; replicate [1]Float%
16Activation at 0.933 uM [2] (0.933μM**)Value of % activation at 0.933 uM; replicate [2]Float%
17Activation at 0.933 uM [3] (0.933μM**)Value of % activation at 0.933 uM; replicate [3]Float%
18Activation at 0.309 uM [1] (0.309μM**)Value of % activation at 0.309 uM; replicate [1]Float%
19Activation at 0.309 uM [2] (0.309μM**)Value of % activation at 0.309 uM; replicate [2]Float%
20Activation at 0.309 uM [3] (0.309μM**)Value of % activation at 0.309 uM; replicate [3]Float%
21Activation at 0.102 uM [1] (0.102μM**)Value of % activation at 0.102 uM; replicate [1]Float%
22Activation at 0.102 uM [2] (0.102μM**)Value of % activation at 0.102 uM; replicate [2]Float%
23Activation at 0.102 uM [3] (0.102μM**)Value of % activation at 0.102 uM; replicate [3]Float%
24Activation at 0.0347 uM [1] (0.0347μM**)Value of % activation at 0.0347 uM; replicate [1]Float%
25Activation at 0.0347 uM [2] (0.0347μM**)Value of % activation at 0.0347 uM; replicate [2]Float%
26Activation at 0.0347 uM [3] (0.0347μM**)Value of % activation at 0.0347 uM; replicate [3]Float%
27Activation at 0.0115 uM [1] (0.0115μM**)Value of % activation at 0.0115 uM; replicate [1]Float%
28Activation at 0.0115 uM [2] (0.0115μM**)Value of % activation at 0.0115 uM; replicate [2]Float%
29Activation at 0.0115 uM [3] (0.0115μM**)Value of % activation at 0.0115 uM; replicate [3]Float%
30Activation at 0.0038 uM [1] (0.0038μM**)Value of % activation at 0.0038 uM; replicate [1]Float%
31Activation at 0.0038 uM [2] (0.0038μM**)Value of % activation at 0.0038 uM; replicate [2]Float%
32Activation at 0.0038 uM [3] (0.0038μM**)Value of % activation at 0.0038 uM; replicate [3]Float%
33Activation at 0.0013 uM [1] (0.0013μM**)Value of % activation at 0.0013 uM; replicate [1]Float%
34Activation at 0.0013 uM [2] (0.0013μM**)Value of % activation at 0.0013 uM; replicate [2]Float%
35Activation at 0.0013 uM [3] (0.0013μM**)Value of % activation at 0.0013 uM; replicate [3]Float%

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

Data Table (Concise)
Classification
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