Late stage counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 5 (S1P5)
Name: Counterscreen assay for S1P4 antagonists: Fluorescence dose response cell-based screening assay for antagonists of the Sphingosine 1-Phosphate Receptor 5 (S1P5) ..more
BioActive Compounds: 3
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: S1P5_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 5 (S1P5)
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, Sphingosine 1 Phosphate Receptor 5, S1PR5, S1P5, Endothelial differentiation gene 8, EDG8, counterscreen, BLA, beta-lactamase, Tango, GPCR, fluorescence, antagonist, inhibitor, HTS, 384, Scripps, Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to identify compounds that act as antagonists of S1P5. This assay uses Tango S1P5-BLA U2OS cells which contain the human Endothelial Differentiation Gene 8 (EDG8; S1P5) 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 S1P5 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 S1P5 antagonists will inhibit S1P5 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 using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 micromolar.
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, and 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
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.
The PubChem Activity Score range for active compounds is 100-61, for inactive 55-0.
List of Reagents:
Tango EDG6-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)
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.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: U-2 OS
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)