کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2511861 1118295 2015 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Forskolin-free cAMP assay for Gi-coupled receptors
موضوعات مرتبط
علوم پزشکی و سلامت داروسازی، سم شناسی و علوم دارویی داروشناسی
پیش نمایش صفحه اول مقاله
Forskolin-free cAMP assay for Gi-coupled receptors
چکیده انگلیسی

G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors.We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described.Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels.In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening.This method will facilitate the study and screening of Gi-coupled receptors for active ligands.

Figure optionsDownload as PowerPoint slide

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemical Pharmacology - Volume 98, Issue 3, 1 December 2015, Pages 381–391
نویسندگان
, , , , , ,