Late-stage counterscreen panel assay for S1P3 agonists: Ricerca HitProfilingScreen + CYP450
Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions in both intracellular (1) and extracellular compartments (2), including a wide variety of physiological responses such as heart rate (3-4), coronary artery caliber, endothelial integrity, and lymphocyte recirculation (4-7). These responses are mediated through high-affinity more ..
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: S1P3_AG_RICERCA HITPROFILING SCREEN_2X%INH
Name: Late-stage counterscreen panel assay for S1P3 agonists: Ricerca HitProfilingScreen + CYP450.
Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions in both intracellular (1) and extracellular compartments (2), including a wide variety of physiological responses such as heart rate (3-4), coronary artery caliber, endothelial integrity, and lymphocyte recirculation (4-7). These responses are mediated through high-affinity interactions with five members of the endothelial differentiation gene (EDG) family of plasma membrane-localized G-protein-coupled receptors (GPCRs), the sphingosine lipid receptors, S1P1-5 (8-10). S1P3 receptor couples promiscuously to Gi, Gq, and G12/13 proteins (11-13). Its expression is widespread (14-16). The S1P3 knockout mouse is phenotypically normal (14). Most S1P-mediated responses on endothelial cells occur via the S1P1 receptor alone or in combination with the S1P3 receptor. Bradycardia and hypertension are clearly associated with S1P3 activation and its expression patterns in cardiac tissue (3, 17). The use of the S1P1-selective agonist SEW2871 together with S1P3-deletant mice showed that activation of S1P3 regulates sinus rhythm, whereas activation of S1P1 plays no discernable role in the process (4). S1P3 on dendritic cells has been identified as a major exacerbating factor for mortality during sepsis by playing a role in the critical linkage of inflammation and coagulation pathways downstream of the thrombin cascade (18). A potent and selective S1P3 agonist would be useful in dissecting the complexities of S1P-mediated physiological processes in which S1P3 is involved, including bradycardia and hypertension.
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sphingosine receptor, Sphingosine-1-phosphate receptor 3, S1P3, endothelial differentiation sphingolipid G-protein-coupled receptor 3, EDG3, agonist, activator, GPCR, counterscreen, panel assay, Ricerca, HitProfilingScreen, Sf9 cells, radioligand binding, late stage, late stage AID, powders, bradycardia, hypertension, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Probe Production Centers Network, MLPCN
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 agonist compound SID 87544119 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.
A response of greater than or equal to 50% inhibition or stimulation is considered active. Negative inhibition represents a stimulation of binding.
List of Reagents:
Reagents were provided by Ricerca Biosciences, LLC.