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 Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: U01 AI074564
Grant Proposal PI: Michael Oldstone, TSRI
External Assay ID: U-2OSCYTOX_INH_LUMI_384_CC50

Name: Late-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.

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 (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, EDG6, LPC1, agonist, activator, luminescence, U-2OS, cytotoxicity, CellTitre-Glo, CC50, late stage, late stage AID, powders, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLSCN

Assay Overview:

The purpose of this assay is to determine cytotoxicity of a powder sample of a compound 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). In this assay, U-2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 uM.

Protocol Summary:

This assay was started by dispensing U-2OS cells in McCoy's 5A medium plus 10% FBS, penicillin 100 U/ml and streptomycin 100 ug/ml (20 uL, 4 x 10E3 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 uM in growth media) were made. 5 uL of diluted compound or media were added to wells, giving final compound concentrations of 0-20 uM. The plate was incubated at 37 C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 uL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader.

The % growth inhibition for each well was then calculated as follows:

% Growth Inhibition = ( MedianRFU_High_Control - RFU_Test_Compound ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).

Percent growth inhibition was plotted against the log of the compound concentration. The CC50 is reported as "> X uM" (where X = the highest concentration tested for which < 50% growth inhibition was observed).

PubChem Activity Outcome and Score:

Compounds with a CC50 value of less than 10uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10uM were considered inactive (non-cytotoxic).

The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

List of Reagents:

U-2OS cells (ATCC, part HTB-96)
McCoy's 5A Medium (Invitrogen, part 16600-082)
FBS (Invitrogen, part 26140-079)
Penicillin / Streptomycin (Invitrogen, part 15140-122)
Cell Titer-Glo (Promega, part G7572)
384-well plates (Corning 3570)

This assay was performed by the assay provider with powder samples of compounds. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.

Grant Number: U01 AI074564