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Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

Cardiovascular Disease Group, Aventis Pharma, 65926 Frankfurt, Germany

Sorin Tunaru

Pharmakologisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany

Andree Blaukat

Pharmakologisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany

Bruce Harris

CNS Disease Group, Aventis Pharmaceuticals, Bridgewater NJ, 08807-0800

Evi Kostenis

Cardiovascular Disease Group, Aventis Pharma, 65926 Frankfurt, Germany, ek{at}7tm.com

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P₅ receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca²⁺ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC₅₀ values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. (Journal of Biomolecular Screening 2003:500-510)

Key Words: G protein-coupled receptor • G protein • sphingosine 1-phosphate • S1P5 receptor

Journal of Biomolecular Screening, Vol. 8, No. 5, 500-510 (2003)
DOI: 10.1177/1087057103257555


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