High-throughput screening with a miniaturized radioligand competition assay identifies new modulators of human ¦Á₂-adrenoceptors

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• 作者：Adyary Fallarero^a ; ¹ ; Katariina Pohjanoksa^b ; ¹ ; Gloria Wissel^c ; Ulla-Mari Parkkisenniemi-Kinnunen^a ; Henri Xhaard^c ; Mika Scheinin^b ; Pia Vuorela^a ; ^{pia.vuorela@abo.fi}
• 关键词：¦Á2-Adrenoceptors ; Lysergol ; 2&Prime ; 2?&prime ; -Bisepigallocatechin digallate ; Partial agonist ; GPCR ; Chemical libraries
• 刊名：European Journal of Pharmaceutical Sciences
• 出版年：2012
• 出版时间：18 December, 2012
• 年：2012
• 卷：47
• 期：5
• 页码：941-951
• 全文大小：1102 K

文摘

Human ¦Á₂-adrenoceptors (¦Á₂-ARs) are rhodopsin-like G-protein coupled receptors, and potential drug targets. The three different human ¦Á₂-AR subtypes ¦Á_2A, ¦Á_2B and ¦Á_2C are widely distributed in tissues, but so far only a few subtype-selective ligands have been identified. In this project, we set off to conduct a large chemical screen for activity on the human ¦Á_2B-AR and studied the selectivity of the active compounds towards the human ¦Á_2A- and ¦Á_2C-AR subtypes. We employed a radioligand competition binding assay that was optimized and miniaturized into a robotic environment. Membrane fractions containing recombinant human receptor subtypes were prepared from stably transfected Chinese hamster ovary (CHO) cell lines. Initially identified hits were followed up and characterized, and chemoinformatics tools were applied to gain better understanding of the relevance of the results. After a primary screen against ¦Á_2B-AR, 176 compounds of the 17,952 included in the library were declared as active at 10 ¦ÌM, of which 89 compounds were further selected for potency and affinity determinations using the three human ¦Á₂-AR subtypes. One of the identified positive hits was , which was found to have high affinity at all three human ¦Á₂-AR subtypes. This represents the first non-protonable molecule identified as able to interact with these receptors. Additionally, results obtained with a functional assay (agonist-induced stimulation of [³⁵S]GTP¦ÃS binding) supported the identification of another positive hit, lysergol, as a partial agonist of the human ¦Á₂-AR subtypes. The dataset of confirmed active chemical species represents a readily available, high quality source for follow-up studies. Altogether, these results provide novel research approaches for drug discovery of modulators of the ¦Á₂-AR subtypes.