Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
ACS Chem Biol. 2012 May 18;7(5):901-10. doi: 10.1021/cb200360z. Epub 2012 Mar 12.
Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and β-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of β2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.
已经开发出用于评估潜在治疗性试剂对 G 蛋白偶联受体 (GPCR) 功效的方法。此前，我们使用 96 孔微板和基于分裂萤火虫荧光素酶互补的生物发光显微镜，展示了对 GPCR 和β-抑制蛋白 2 (ARRB2) 相互作用的敏感检测。在此，我们使用发射更长波长光的萤火虫荧光素酶，在 96 孔板测定中进行单细胞成像，对β2-肾上腺素能受体 (ADRB2) 与 ARRB2 的相互作用进行定量分析。此外，我们展示了在两种系统中进行的 ADRB2-ARRB2 相互作用的生物发光体内成像：细胞植入和水力尾静脉 (HTV) 方法。具体来说，在 HTV 方法中，在完整的小鼠系统中，通过刺激 ADRB2 的激动剂获得了来自肝脏的发光信号。结果表明，该方法能够在体内测试中对特定器官的化学物质功效进行非侵入性筛选。这种体内系统有助于对药代动力学和药效动力学进行有效评估，并加速 GPCR 新药的开发。
