Cisbio Bioassays, R&D, Parc Marcel Boiteux, BP 84175, 30200 Codolet , France +33 0 466796769 ;
Expert Opin Drug Discov. 2011 Oct;6(10):981-94. doi: 10.1517/17460441.2011.608658. Epub 2011 Sep 8.
G-protein-coupled receptors (GPCRs) are transmembrane proteins that play a key role in the signal transduction of extracellular stimuli. GPCRs associate to a complex assembly of intracellular proteins regulating a large variety of signaling pathways. In particular, the production of inositol 1,4,5 triphosphate (IP3) signs the activation of Gq-coupled receptors. However, its very short half-life makes its assessment too challenging for drug screening operations and the monitoring of calcium release, triggered by IP3, has been extensively used as a downstream readout of this signaling pathway. Recently, a new homogeneous time-resolved fluorescence (HTRF) assay, detecting a downstream metabolite of IP3, inositol monophosphate (IP1), has overcome the drawbacks of the IP3 quantification, allowing its use in primary or secondary screening.
This review provides an overview of the use of the IP-One assay in screening processes, providing comparisons with results obtained with other existing techniques traditionally used to investigate Gq-coupled receptors. Moreover, the review highlights two key features of the IP-One assay, the discrimination of slow acting compounds and the characterization of inverse agonists, which are impossible to achieve using calcium release.
The IP-One assay is well established to perform screening in the pharmaceutical industry. A number of criteria can be taken into account, including the impact of the sensitivity improvement of the assay, to position the IP-One assay in the different stages of the drug screening process. Moreover, the IP-One assay can be used as a valuable solution to investigate new research concepts such as ligand-biased signaling or receptor heteromerization.
G 蛋白偶联受体（GPCRs）是一种跨膜蛋白，在细胞外刺激的信号转导中起着关键作用。GPCRs 与调节多种信号通路的细胞内蛋白质复合物相关联。特别是，三磷酸肌醇（IP3）的产生标志着 Gq 偶联受体的激活。然而，其半衰期非常短，使得其在药物筛选操作中的评估变得极具挑战性，并且 IP3 引发的钙释放已被广泛用作该信号通路的下游读出。最近，一种新的均相时间分辨荧光（HTRF）测定法，检测 IP3 的下游代谢物肌醇单磷酸（IP1），克服了 IP3 定量的缺点，允许其在初级或次级筛选中使用。
本文综述了 IP-One 测定法在筛选过程中的应用，与传统用于研究 Gq 偶联受体的其他现有技术的结果进行了比较。此外，该综述强调了 IP-One 测定法的两个关键特征，即对缓慢作用化合物的区分和反向激动剂的特性，这是使用钙释放无法实现的。
IP-One 测定法已在制药行业中广泛用于筛选。可以考虑许多标准，包括测定法灵敏度提高的影响，以将 IP-One 测定法定位在药物筛选过程的不同阶段。此外，IP-One 测定法可用作研究新研究概念（如配体偏向信号或受体异源二聚化）的有价值的解决方案。
