Counterscreen panel assay for S1P4 antagonists: Ricerca HitProfilingScreen + CYP450
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 more ..
Late-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 compounds
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: S1P4_ANT_RICERCA HITPROFILING SCREEN_%INH
Name: Counterscreen panel assay for S1P4 antagonists: Ricerca HitProfilingScreen + CYP450
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, Endothelial differentiation gene 6, EDG6, counterscreen, antagonist, inhibitor, Ricerca, HitProfilingScreen, Sf9 cells, radioligand binding, Scripps, Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Assay Overview: The purpose of this panel of binding assays performed by Ricerca Biosciences, LLC, is to identify a subset of potential receptors, transporters, ion channels, etc. for which the S1P4 antagonist compound SID 87357351 displays affinity.
Assays for CYP450, 1A2; CYP450, 2C19; CYP450, 2C9; CYP450, 2D6; and CYP450, 3A4 were enzyme assays using human recombinant insect Sf9 cells with 5 uM 3-cyano-7-ethoxycoumarin as substrate (except for CYP450, 3A4, which used 50 uM 7-benzyloxy-4-(trifluoromethyl)-coumarin as substrate). Detection was based on spectrofluorimetric quantitation of the enzymatic product produced. Assays for the other targets were radioligand binding assays.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
A response of >= 50% inhibition or stimulation is considered "active". Negative inhibition represents a stimulation of binding.
List of Reagents:
Reagents were provided by Ricerca Biosciences, LLC.
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