Ehlert Frederick J, Griffin Michael T
Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, CA 92617-4625, United States; Crean School of Health and Life Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA, United States.
Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, CA 92617-4625, United States; Crean School of Health and Life Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA, United States.
J Pharmacol Toxicol Methods. 2014 May-Jun;69(3):253-79. doi: 10.1016/j.vascn.2014.01.002. Epub 2014 Jan 13.
The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants.
Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands - including inverse agonists - and the efficacy of the unoccupied receptor (i.e., constitutive activity).
We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration-response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor.
配体与受体活性和非活性状态的亲和常数最终决定其激活下游信号事件的能力。在本报告中，我们描述了一种用于估计这些微观常数的逆向工程策略。
我们的方法包括在变构调节以及受体表达降低或部分受体失活的条件下，分析G蛋白偶联受体信号通路中下游测量的反应。该分析还得出未占据受体的异构化常数、信号通路的敏感性常数以及受体群体的更多经验参数，包括变构和正构配体（包括反向激动剂）的观察到的亲和力和效力以及未占据受体的效力（即组成性活性）。
我们通过分析证明和模拟数据分析验证了我们的方法。我们还使用我们的方法分析文献数据。我们表明，只要相同的活性状态介导反应，正构和变构配体的微观常数的值与变构相互作用和受体 - 信号通路的性质无关。我们的分析对于量化探针依赖性变构相互作用和激动剂对不同信号通路的选择性很有用。了解给定细胞系或组织制剂中信号通路的异构化常数和敏感性常数，使未来的研究人员能够通过分析其浓度 - 反应曲线简单地估计激动剂对受体状态的亲和常数。我们的方法还提供了一种验证受体活性和非活性状态晶体结构的配体亲和力的计算机模拟估计的方法。
