PROJECT2:Understanding of Functionally-Selective D2 Dopamine Receptor Ligands

项目2：功能选择性 D2 多巴胺受体配体的理解

• 批准号: 7623085
• 负责人: Richard B Mailman
• 金额: $ 25.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623085
• 关键词: Adenylate Cyclase; Affect; Agonist; Animals; Arachidonic Acids; Arts; Attenuated; Behavior; Behavioral; Behavioral Assay; Behavioral Model; Binding; Biological Assay; Cell Line; Cells; Characteristics; Classification; Clinical; Development; Dopamine; Dopamine Agonists; Dopamine D2 Receptor; End Point; Foundations; Goals; Guanosine Triphosphate Phosphohydrolases; Hand; Human; In Situ; In Vitro; Knowledge; Lead; Libraries; Ligands; Link; Mediating; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular Conformation; Mus; Mutation; Neuroglia; Neurons; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Primates; Property; Protein Isoforms; Psychopathology; Public Domains; Purpose; Quinpirole; Rate; Receptor Signaling; Relative (related person); Research; Research Design; Schizophrenia; Screening procedure; Short-Term Memory; Signal Pathway; Signal Transduction; Site; Structure; Symptoms; System; Therapeutic; Tissues; Work; aripiprazole; base; concept; dopamine D2L receptor; dopaminergic neuron; drug discovery; high throughput analysis; in vivo; interest; novel; pre-clinical; receptor; tool

Project #2. Discovery and mechanistic understanding of functionally-selective D2 dopamine receptor ligands (Richard Mailman, Project PI) A. SPECIFIC AIMS The idea that some ligands can selectively activate only some of the signaling pathways mediated by a single receptor isoform ("functional selectivity") is a novel concept that has markedly changed classic concepts of receptor pharmacology. During the past few years, there has been overwhelming support for this concept from more than a dozen receptor systems. We, and almost all of the other labs who have worked in this arena, have recognized that it has profound implications for drug discovery. As discussed in the overall introduction to this application, the functional selectivity hypothesis predicts that functionally selective drugs (i.e., those that differentially activate (or inhibition) signaling pathways linked to a single receptor) will be differentiated not only by their behavior in vitro, but that this will lead to different behavioral and physiological effects seen both preclinically and clinically. This project will focus on the dopamine D2L receptor, and utilize a battery of signaling assays which are independent (or largely independent) of each other. These assays allow the differential classification of functionally selective properties, and allow the rational selection of agents with novel properties. These assays, as well as new assays in development, will allow for the discovery and rational classification of novel functionally selective dopamine agonists. Novel leads identified from in vitro screening campaigns will be profiled computationally and physically for off-target actions and suitably selective compounds will subsequently be evaluated in a variety of behavioral assays that will offer clues predictive of potential therapeutic action. We recognize that the current state-of-the-art is not adequately advanced such that it is possible to predict from a novel pattern of signaling activity seen in vitro whether the candidate ligand might have clinical superiority in discrete domains of psychopathology. In this regard, however, the examination of interesting candidate compounds both in traditional behavioral models and in mice with specific genetic alterations will provide a powerful way to select compounds that might become drug candidates. As an example, one inherent hypothesis is that functionally selective drugs can be discovered that cause significant facilitation of dopaminergic functions that modulate working memory, while still effectively attenuating dopaminergic function that decreases positive symptoms of schizophrenia. The foundation of research from the past two decades makes us believe that we can confirm this optimistic hypothesis. We shall pursue this via the following aims that are heavily integrated with Projects 1 and 3, and will make use of both scientific cores. 1. AIM 1. CHARACTERIZE EXISTING AND DISCOVERY COMPOUNDS FOR FUNCTIONALLY SELECTIVE PROPERTIES IN NON-NEURONAL D2L DOPAMINE RECEPTOR SYSTEMS Hypothesis: A battery of signaling assays that are independent or largely independent of each other will differentiate drugs with functionally selective properties, and allow selection of agents with novel properties. Corollary: The pattern of signaling effects will differentiate the ligands one from the other and be predictive of preclinical behavioral and clinical differences, although not necessary clinical superiority. The goal is to discover novel functionally selective D2 signalers that can provide new lead molecules, and/or ligands that may be useful research tools. The broad array of existing D2L functional assays are expressed in non-neuronal cell lines (CHO and HEK). Although it would be ideal to know the exact mechanisms about how the D2L receptor signals in situ in primate tissue, this knowledge does not currently exist (although it will be explored in Project 1. On the other hand, the current battery can discriminate functionally selective D2 ligands that have unique behavioral properties in animals and humans, providing reasonable evidence of validity. We shall focus on relatively small subsets of rationally selected compounds from our Wyeth coinvestigators, from compounds from our recent research, from promising targets we have found in the public domain, and from selected structures available in commercial libraries predicted from our computational approaches. Reference compounds will be dopamine and two "typical" D2L agonists (quinpirole and RNPA), as well as known functionally selective compounds with different patterns of activity (e.g., aripiprazole and S-3- PPP). The compounds will be analyzed in four receptor-mediated functional assays (inhibition of adenylate cyclase, stimulation of MAPK kinase, arachidonic acid release, and stimulation of GTPase binding). Emax and ED50 values will be determined, and functionally selective ligands for detailed study by this Project and Projects 2 and 3 will be those in which there is greater than a 30% difference intrinsic activity in one or more functional endpoints versus the others, or when the ED50 changes by more than ten-fold relative to the reference compounds. Finally, these assays are currently semi-automated, and provide adequate throughput for the focused types of studies we are making now. In parallel, we shall make efforts to increase their throughput rate (without decreasing either accuracy or precision), such that they can be applied to higher throughput analysis for the D2i_ and other receptors. 2. AIM 2: DETERMINATION OF THE STRUCTURAL BASIS FOR ACTIVATION AND FUNCTIONAL SELECTIVITY OF D2L RECEPTORS. Our hypothesis that functional selectivity results from the ligand-unique sets of induced conformations (rather than selection of discrete active states) makes it important to understand some of the structural determinants that are involved. Thus, we hypothesize that functional selectivity can result either from conformational perturbation of the D2|_ receptor done by ligands "sterically" (close to the residues that bind dopamine) and/or "allosterically" (involving both the steric sites and aspects of the receptor not normally engaged by dopamine). This aim will elucidate some of the subtle structural interactions that differentiate how non-selective and functionally selective ligands affect the D2i_ receptor, and how this results in selective activation of specific effector pathways. Computationally-predicted mutations of the D2|_ receptor will be made, and ligand analysis conducted using rigid or semi-rigid compounds that minimize possible clocking poses, as well as functionally selective ligands emerging from Aim 1. 3. AIM 3. DEVELOP NEURONALLY-RELEVANT CELL SPECIFIC ASSAY SYSTEMS Project #1 seeks to develop physiologically relevant ex vivo models for screening for functionally selective drugs. In parallel, we wish to find immortal cell lines that functionally mirror dopamine neurons. Prior work with the MN9D cell line has shown that these cells provide excellent predictability on which functionally selective drugs produce novel behavioral characteristics in vivo. Unfortunately, we have found this like to be unstable, and seek a line that can replace it. We have identified two excellent candidates for our purposes (N27 and CAD), and we shall first characterize these lines for appropriate phenotype, and for their stability when molecularly-manipulated.

项目#2。功能选择性的发现和机制理解 D2 多巴胺受体配体（Richard Mailman，项目 PI） A. 具体目标 一些配体只能选择性地激活某些由配体介导的信号传导途径的想法 单一受体亚型（“功能选择性”）是一个新概念，显着改变了经典概念 受体药理学。在过去的几年里，这个概念得到了压倒性的支持 来自十多个受体系统。我们以及几乎所有在这个领域工作过的其他实验室， 已经认识到它对药物发现具有深远的影响。正如总体介绍中所讨论的 在本申请中，功能选择性假说预测功能选择性药物（即那些 与单个受体相关的差异激活（或抑制）信号通路）不仅会被区分 通过它们在体外的行为，但这将导致不同的行为和生理效应 临床前和临床。该项目将重点关注多巴胺 D2L 受体，并利用一组 彼此独立（或很大程度上独立）的信号传导测定。这些测定允许 功能选择性特性的差异分类，并允许合理选择具有 新颖的特性。这些测定以及正在开发的新测定将允许发现和 新型功能选择性多巴胺激动剂的合理分类。从体外鉴定出的新先导化合物 筛选活动将通过计算和物理方式进行分析，以发现脱靶行为并进行适当的选择性 随后将在各种行为测定中对化合物进行评估，这些测定将提供预测的线索 潜在的治疗作用。 我们认识到，当前的最先进技术还没有足够先进，因此不可能 从体外观察到的信号活动的新模式预测候选配体是否可能具有临床作用 在精神病理学的离散领域具有优势。然而，在这方面，有趣的研究 传统行为模型和具有特定基因改变的小鼠中的候选化合物将 提供了一种有效的方法来选择可能成为候选药物的化合物。举个例子，一个固有的 假设是可以发现功能选择性药物，从而显着促进 调节工作记忆的多巴胺能功能，同时仍有效减弱多巴胺能功能 减少精神分裂症的阳性症状。过去二十年的研究基础 让我们相信我们可以证实这个乐观的假设。我们将通过以下目标来实现这一目标 与项目 1 和 3 高度集成，并将利用这两个科学核心。 1. 目标 1. 表征现有和发现的化合物的功能 非神经元 D2L 多巴胺受体系统的选择性特性 假设：一组相互独立或很大程度上相互独立的信号传导检测将 区分具有功能选择性特性的药物，并允许选择具有新特性的药物。 推论：信号传导效应的模式将区分配体和其他配体，并可预测 临床前行为和临床差异，尽管不一定具有临床优越性。目标是 发现新的功能选择性 D2 信号分子，可以提供新的先导分子和/或配体 成为有用的研究工具。现有的广泛 D2L 功能测定在非神经元细胞中表达 线（CHO 和 HEK）。尽管了解 D2L 受体如何发挥作用的确切机制是理想的 灵长类组织中的原位信号，目前尚不存在这种知识（尽管将在项目中进行探索） 1. 另一方面，当前的电池可以区分具有独特功能的功能选择性 D2 配体 动物和人类的行为特性，提供合理的有效性证据。 我们将重点关注惠氏联合研究人员合理选择的化合物的相对较小的子集， 来自我们最近研究的化合物，来自我们在公众中发现的有希望的目标 域，以及从我们的计算预测的商业图书馆中可用的选定结构中 接近。参考化合物是多巴胺和两种“典型”D2L 激动剂（喹吡罗和 RNPA），如 众所周知的具有不同活性模式的功能选择性化合物（例如阿立哌唑和 S-3- 购买力平价）。这些化合物将在四种受体介导的功能测定中进行分析（抑制腺苷酸 环化酶、MAPK 激酶刺激、花生四烯酸释放和 GTP 酶结合刺激）。 Emax 和 将确定 ED50 值，并通过该项目和功能选择性配体进行详细研究 项目 2 和 3 的一项或多项内在活动差异大于 30% 功能终点与其他终点相比，或者当 ED50 相对于其他终点变化超过十倍时 参考化合物。 最后，这些测定目前是半自动化的，并为重点关注提供足够的通量。 我们现在正在进行的研究类型。与此同时，我们将努力提高他们的吞吐率（不 降低准确度或精确度），这样它们就可以应用于更高通量的分析 D2i_ 和其他受体。 2. 目标 2：确定激活和功能的结构基础 D2L 受体的选择性。 我们的假设是功能选择性是由配体独特的诱导构象组产生的 （而不是选择离散的活性状态）使得理解一些结构变得很重要 所涉及的决定因素。因此，我们假设功能选择性可能来自于 由配体“空间”（靠近结合的残基）对 D2|_ 受体进行构象扰动 多巴胺）和/或“变构”（涉及受体的空间位点和方面，通常不 多巴胺参与）。这一目标将阐明一些微妙的结构相互作用，这些相互作用区分了如何 非选择性和功能选择性配体影响 D2i_ 受体，以及这如何导致选择性 特定效应途径的激活。将产生 D2|_ 受体的计算预测突变， 和使用刚性或半刚性化合物进行配体分析，最大限度地减少可能的时钟姿势，如 以及目标 1 中出现的功能选择性配体。 3. 目标 3. 开发神经相关细胞特异性检测系统 项目#1 寻求开发生理相关的离体模型来筛选功能性 选择性药物。与此同时，我们希望找到功能上反映多巴胺神经元的永生细胞系。事先的 与 MN9D 细胞系的合作表明，这些细胞提供了极好的可预测性 选择性药物在体内产生新的行为特征。不幸的是，我们发现这就像 不稳定，寻找可以替代它的线路。我们已经为我们的目的确定了两名优秀的候选人 （N27 和 CAD），我们将首先表征这些品系的适当表型及其稳定性 当分子操纵时。