Pharmacodynamics of Biased G protein-Coupled Receptor Agonism

描述（由申请人提供）：偏倚G蛋白偶联受体（GPCR）激动剂是具有途径选择性功效的正位配体，仅激活或抑制其同源受体的一部分信号库。与通过改变受体效力的量来起作用的传统激动剂不同，偏倚激动剂具有定性改变信号传导的能力，这表明利用配体偏倚来开发最大化临床疗效同时最小化副作用的药物是可能的。我们发现1型甲状旁腺激素受体（PTH1R）的抑制素途径选择性偏倚激动剂促进体内合成代谢骨形成，而不刺激骨吸收或产生高钙血症，这一发现为偏倚激动剂可用于引发传统激动剂或拮抗剂无法实现的生物反应提供了主要证据。利用生物信息学代谢途径和基因聚类分析，我们发现抑制素选择性PTH1R激动剂在组织水平上产生独特的基因组“足迹”，即其作用机制与传统激动剂不同，并且无法根据经典GPCR信号传导的现有知识进行预测。这一关键观察结果提出了关于偏倚激动作用性质的基本问题，我们建议在本应用中解决这一问题。我们将采用体外配体功效分析、基于微阵列的功能基因组分析和基于细胞的生物反应分析来解决三个特定目标。目的1将使用野生型和-拘捕素2-/-小鼠的初代颅骨成骨细胞和PTH1R配体来确定普通细胞类型中常规、G蛋白途径选择性和拘捕素途径选择性激动剂的功能特征。我们假设G蛋白选择性和抑制蛋白选择性偏向激动剂都会产生与传统激动剂在质量上不同的基因组足迹。目的2将利用原代肾小管上皮细胞和血管紧张素AT1AR、血管加压素V2R和PTH1R的选择性偏配体来确定在共同细胞背景下通过不同的gpcr激活阻滞素信号通路的效果。我们假设对不同的抑制选择性激动剂的反应会重叠，而对常规激动剂的反应会因同时激活不同的G蛋白而产生分歧。目的3将利用原代成骨细胞、肾小管上皮细胞和心肌细胞，以及AT1AR和PTH1R的阻滞蛋白通路选择性偏配体，来确定通过通用GPCR激活阻滞蛋白信号通路在不同细胞类型中的作用。我们假设，在不同的细胞背景下，抑制素信号的激活将产生类似的效果，这反映了有限的抑制素依赖信号库。这项工作将确定影响的范围

DESCRIPTION (provided by applicant): Biased G protein-coupled receptor (GPCR) agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. Unlike conventional agonists that work by changing the quantity of receptor efficacy, biased agonists possess the ability to qualitatively change signaling, suggesting it may be possible to exploit ligand bias to develop drugs that maximize clinical effectiveness while minimizing side effects. Our finding that an arrestin pathway-selective biased agonist for the type 1 parathyroid hormone receptor (PTH1R) promotes anabolic bone formation in vivo without stimulating bone resorption or producing hypercalcemia offers proof of principal that biased agonists can be used to elicit biological responses that cannot be achieved with conventional agonist or antagonist drugs. Using bioinformatic metabolic pathways and gene cluster analysis, we found that the arrestin-selective PTH1R agonist produces a unique genomic 'footprint' at the tissue level, i.e. that its mechanism of action is distinct from that of a conventional agonist and could not have been predicted on the basis of existing knowledge of classical GPCR signaling. This key observation raises fundamental questions about the nature of biased agonism, which we propose to address in this application. We will employ in vitro ligand efficacy profiling, microarray-based functional genomic analyses, and cell-based assays of biological response, to address three Specific Aims. Aim 1 will employ primary calvarial osteoblasts from wild type and ¿-arrestin2-/- mice and PTH1R ligands to determine the functional characteristics of conventional, G protein pathway-selective, and arrestin pathway-selective agonists in a common cell type. We hypothesize that both G protein-selective and arrestin-selective biased agonists will produce genomic footprints that are qualitatively different from a conventional agonist. Aim 2 will employ primary renal tubular epithelial cells and arrestin pathway- selective biased ligands for the angiotensin AT1AR, vasopressin V2R, and PTH1R to determine the effects of activating arrestin signaling pathways via different GPCRs in a common cell background. We hypothesize that the response to different arrestin-selective agonists will overlap, whereas the responses to conventional agonists will diverge due to the concomitant activation of different G proteins. Aim 3 will employ primary osteoblasts, renal tubular epithelial cells and cardiomyocytes, and arrestin pathway-selective biased ligands for the AT1AR and PTH1R to determine the effects of activating arrestin signaling pathways via a common GPCR in different cell types. We hypothesize that activation of arrestin signaling will produce similar effects in different cell backgrounds, reflecive of a limited arrestin-dependent signaling repertoire. This work will establish the range of effects that can be achieved using arrestin-selective agonists, and determine the extent to which arrestin-dependent effects are conserved across different GPCRs and target tissues. This information will be critical to efforts to develop novel therapeutics that exploit ligand bias.