Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 2 (S1P2)
Name: Counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 2 (S1P2) ..more
BioActive Compounds: 5
Depositor Specified Assays
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: S1P2_ANT_BLA_384_3XIC50
Name: Counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 2 (S1P2)
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.
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, S1PR4, EDG6, Sphingosine-1-phosphate receptor 2, S1P2, S1PR2, GPCR, betalactamase, reporter gene, endothelial differentiation, antagonist, inhibitor, HTS, counterscreen, 384, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN
A Chinese Hamster Ovary (CHO) cell line stably transfected with the human S1P2 receptor and a cAMP Response Element-beta lactamase (CRE-BLA) reporter construct was used to measure S1P2 antagonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. In this assay, cells have been stimulated with the S1P agonist, which stimulates the S1P2 receptor, hence increasing BLA gene transcription. An antagonist is identified as a compound that prevents activation by exogenously added S1P. Therefore, in the presence of a potential S1P2 antagonist, transcription of the BLA gene will decrease. This decrease is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell permeable BLA substrate.
As designed, test compounds that act as S1P2 antagonists will inhibit BLA transcription and cleavage of the fluorogenic substrate, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 micromolar.
Cells were cultured in T-175 sq cm flasks at 37 deg C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media supplemented with 10% v/v heat inactivated dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 2 mg/mL Geneticin and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). Prior to assay, cells were suspended to a concentration of 1.25 million/milliliter in assay media, which consisted of phenol red-free Dulbecco's Modified Eagle's Media supplemented with 2% charcoal/dextran-treated fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin).
The assay began by dispensing 4 microliters of cell suspension to each test well of a 1536 well plate (5000 cells/well) followed by incubation at 37 deg C in 5% CO2 for 16 hrs. To the appropriate wells were then added 26 nL of test compound in DMSO (final nominal concentration of 5 micromolar, final DMSO concentration of 0.5%) or DMSO only (for high control wells) followed directly afterwards by 1 microliter of S1P in 2% BSA (final concentration of 750 nanomolar, i.e. a concentration that resulted in 80% activity). The high control (EC80 challenge) and low control (100% antagonism) were added to the appropriate control wells and plates were incubated again at 37 deg C in 5% CO2 for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer's protocol and 1 microliter of this mixture was then added to each well. After a further 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and fluorescence emission wavelengths of 535 nm & 460 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
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.
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. 25 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 25 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar 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.
List of Reagents:
Dulbecco's Modified Eagle's Media with phenol red (Invitrogen, part 11965-092)
Dulbecco's Modified Eagle's Media without phenol red (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26400-044)
NEAA (Invitrogen, part 1114-050)
Sodium Pyruvate (Invitrogen, part 11360-070)
HEPES (Invitrogen, part 15630-080)
L-Glutamine (Invitrogen, part 25030-081)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055).
Charcoal/dextran treated fetal bovine serum (Hyclone, part SH30068.03)
Probenicid (Sigma, part P8761)
S1P agonist (Biomol, part SL-140)
LiveBLAzer (Invitrogen, part K1096)
JTE013 (Tocris, part 2392 ) 384-well plates (Greiner, part 788092)
T175 tissue culture flasks (Corning, part 431080)
All data reported were normalized on a per-plate basis. 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)