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BioAssay: AID 1564

Counterscreen assay for S1P4 antagonists: Cell-based high throughput screening assay to identify antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)

Name: Counterscreen assay for S1P4 antagonists: Cell-based high throughput screening assay to identify antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1) ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 1564
Data Source: The Scripps Research Institute Molecular Screening Center (S1P1_ANT_BLA_1536_3X%ACT Counterscreen)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-03-19
Modify Date: 2010-06-15

Data Table ( Complete ):           Active    All
BioActive Compounds: 193
Depositor Specified Assays
1510Primary Cell-Based Assay to Identify Antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)screening
1524Confirmation cell-based high throughput assay for antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)screening
1853Summary of probe development efforts to identify antagonists of Sphingosine 1-Phosphate Receptor 4 (S1P4)summary1
489009Late-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 compoundsconfirmatory
489017Counterscreen panel assay for S1P4 antagonists: Ricerca HitProfilingScreen + CYP450other
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_ANT_BLA_1536_3X%ACT Counterscreen

Name: Counterscreen assay for S1P4 antagonists: Cell-based high throughput screening assay to identify antagonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1)


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 inhibition 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 inhibition of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gαi and Gαo 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.

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.


Sphingosine Receptor, Sphingosine-1-phosphate receptor 4, S1P4, EDG6, LPC1, antagonist, inhibitor, HTS, GPCR, 1536, Tango, FRET, GAL4-VP16, beta-arrestin, beta-lactamase, BLA, reporter gene, fluorescence, counterscreen, S1P1, EDG1, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Probe Production Center Network, MLPCN.
Assay Overview:

The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, "Primary Cell-Based Assay to Identify Antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)" (PubChem AID 1510), and that confirmed activity in a set of experiments entitled, "Confirmation Cell-Based Assay to Identify Antagonists of the Sphingosine 1-Phosphate Receptor 4 (S1P4)" (PubChem AID 1524), were nonselective antagonists due to inhibition 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 antagonists will inhibit S1P1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate at a final nominal concentration of 5 micromolar.

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 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 microliters 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 (10 nM final nominal EC80 concentration) prepared in 2% 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 microliter/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)))
Test_Compound is defined as wells containing test compound and S1P,
Low_Control is defined as wells containing S1P,
High_Control is defined as wells containing DMSO only.

A mathematical algorithm was used to determine nominally inhibiting compounds in the screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed inhibition value. Negative % inhibition values are reported as activity score zero.

The inactive compounds of this assay have activity score range of 0 to 24 and active compounds range of activity score is 24 to 100.

List of Reagents:

Tango EDG-1-bla U2OS cells (Invitrogen, part K1622)
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)
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 4 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
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (4.98μM**)Normalized percent inhibition of the primary screen at a compound concentration of 4.98 micromolar.Float%
2Standard DeviationStandard deviation of the confirmation screen derived from the normalized percent inhibition of the triplicate data for each compound.Float

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

Data Table (Concise)