Pharmacodynamics of Biased G protein-Coupled Receptor Agonism

描述（由申请人提供）：偏置G蛋白偶联受体（GPCR）激动剂是具有路径选择性功效的正构配体，仅激活或抑制其同源受体的信号库的子集。与通过改变受体功效的量起作用的常规激动剂不同，偏性激动剂具有定性改变信号传导的能力，这表明可能利用配体偏性来开发最大化临床有效性同时最小化副作用的药物。我们的发现是，1型甲状旁腺激素受体（PTH 1 R）的抑制蛋白途径选择性偏向激动剂可促进体内合成代谢骨形成，而不会刺激骨吸收或产生高钙血症，这提供了原则证据，即偏向激动剂可用于引发生物学反应，而传统激动剂或拮抗剂药物无法实现。使用生物信息学代谢途径和基因簇分析，我们发现抑制蛋白选择性PTH 1 R激动剂在组织水平产生独特的基因组“足迹”，即其作用机制与传统激动剂不同，并且不能基于经典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有偏激动作用性质的基本问题，我们建议在本申请中解决这些问题。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学反应测定，以解决三个特定目标。目的1将采用野生型和β-arrestin 2-/-小鼠的原代颅骨成骨细胞和PTH 1 R配体，以确定常规的G蛋白通路选择性和arrestin通路选择性激动剂在常见细胞类型中的功能特征。我们假设G蛋白选择性和抑制蛋白选择性偏向性激动剂将产生与常规激动剂在性质上不同的基因组足迹。目的2将采用原代肾小管上皮细胞和血管紧张素AT 1AR、加压素V2 R和PTH 1 R的抑制蛋白途径选择性偏向配体，以确定在共同细胞背景中通过不同GPCR激活抑制蛋白信号传导途径的作用。我们假设，不同的arrestin选择性激动剂的反应将重叠，而传统的激动剂的反应将分歧，由于不同的G蛋白的伴随激活。目的3将使用原代成骨细胞、肾小管上皮细胞和心肌细胞，以及AT 1AR和PTH 1 R的arrestin通路选择性偏置配体，以确定通过共同的GPCR激活arrestin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，抑制蛋白信号的激活将产生类似的效果，尽管抑制蛋白依赖的信号库有限。这项工作将确定影响范围 这可以使用抑制蛋白选择性激动剂实现，并确定抑制蛋白依赖性效应在不同GPCR和靶组织中保守的程度。这一信息将是至关重要的努力，开发新的疗法，利用配体的偏见。

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.