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: 8079092
• 负责人: Richard B Mailman
• 金额: $ 27.11万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079092
• 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; Dopamine Receptor; 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; Quinpirole; Receptor Signaling; Relative (related person); Research; Research Design; Research Personnel; Schizophrenia; Screening procedure; Short-Term Memory; Signal Pathway; Signal Transduction; Site; Structure; Symptoms; System; Therapeutic; Tissues; Work; aripiprazole; base; dopamine D2L receptor; dopaminergic neuron; drug candidate; 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。发现和机械理解功能选择性 D2多巴胺受体配体（Richard Mailman，Project PI） A.具体目标 某些配体可以选择性地激活某些由A介导的信号通路的想法 单个受体同工型（“功能选择性”）是一个新颖的概念，已显着改变了经典概念 受体药理学。在过去的几年中，对这个概念有压倒性的支持 来自十多个受体系统。我们以及几乎所有在这个领域工作的实验室， 已经认识到它对药物发现具有深远的影响。正如在总体介绍中所讨论的 此应用，功能选择性假设预测功能性选择性药物（即 与单个受体相关的差异激活（或抑制）信号通路）不仅会分化 通过他们的体外行为，但这将导致不同的行为和生理影响 临床上和临床上。该项目将重点放在多巴胺D2L受体上，并利用一组 彼此独立（或大部分独立）的信号传导测定。这些测定允许 功能性选择性属性的差异分类，并允许具有合理选择的代理选择 新型特性。这些测定以及开发的新测定法将允许发现和 新型功能性多巴胺激动剂的合理分类。从体外鉴定出的新型潜在客户 筛选活动将在计算和身体上进行介绍，以进行脱离目标，并适当选择性 随后将在各种行为测定中评估化合物，以提供可预测的线索 潜在的治疗作用。 我们认识到，当前的最新目前没有足够的先进，因此有可能 从在体外看到的新型信号传导活性模式预测，候选配体是否可能具有临床 精神病学领域的优势。然而，在这方面，有趣的检查 传统行为模型和具有特定遗传改变的小鼠中的候选化合物都将 提供一种有力的方法来选择可能成为候选毒品的化合物。例如，一个固有的 假设是可以发现功能选择性药物，引起大量促进 多巴胺能调节工作记忆的功能，同时仍有效地衰减多巴胺能功能 这降低了精神分裂症的积极症状。过去二十年来研究的基础 让我们相信我们可以确认这一乐观的假设。我们将通过以下目标来追求这一点 与项目1和3的大量集成在一起，并将利用这两个科学核心。 1。AIM1。表征现有的现有和发现化合物的功能 非神经元D2L多巴胺受体系统的选择性特性 假设：一系列独立或在很大程度上独立的信号传导测定 用功能性选择性的特性区分药物，并允许选择具有新颖性能的药物。 推论：信号效应的模式将使一个配体从另一个区分，并预测 临床前行为和临床差异，尽管不是必需的临床优势。目标是 发现可以提供新的铅分子和/或可能的配体的新颖功能选择性D2信号器 成为有用的研究工具。现有的D2L功能分析的广泛阵列在非神经元细胞中表达 线（Cho和Hek）。尽管了解D2L受体的确切机制是理想的选择 在灵长类组织中原位信号，目前不存在这些知识（尽管将在项目中探讨它 1。另一方面，当前的电池可以区分具有独特的功能性D2配体 动物和人类的行为特性提供了合理的有效性证据。 我们将重点关注从我们的Wyeth共同评估器中相对较小的合理选择化合物的子集，即 从我们最近的研究中的化合物中，从我们在公众中发现的有希望的目标来看 域，以及从我们的计算中预测的商业库中可用的选定结构 方法。参考化合物将是多巴胺和两种“典型” D2L激动剂（奎因螺旋和RNPA） 以及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-- PPP）。这些化合物将在四个受体介导的功能测定中进行分析（抑制腺苷酸盐 循环酶，MAPK激酶的刺激，花生四烯酸释放以及GTPase结合的刺激）。 emax和 将确定ED50值，并在功能上选择性配体，以供该项目详细研究 项目2和3将是一个或多个固有活动差异30％的项目 功能性终点与其他端点，或者相对于ED50变化超过十倍时 参考化合物。 最后，这些测定当前是半自动的，并为专注的 我们现在正在进行的研究类型。同时，我们将努力提高其吞吐率（没有 降低精度或精度），以便将它们应用于更高的吞吐量分析 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从配体唯一诱导构象的集合产生 （而不是选择离散活性状态）使了解一些结构的重要 涉及的决定因素。因此，我们假设功能选择性可以从 D2 | _受体的构象扰动是由配体“静置”（接近绑定的残基）完成的 多巴胺）和/或“变构”（涉及受体的空间位点和方面通常不正常 由多巴胺参与）。这个目标将阐明一些微妙的结构相互作用，以区分 非选择性和功能选择性配体会影响D2I_受体，以及这如何导致选择性 特定效应途径的激活。将制作D2 | _的计算预测的突变， 使用刚性或半刚性化合物进行的配体分析，以最大程度地减少可能的时钟姿势 以及功能上选择性配体从AIM 1出现。 3。目标3。开发与神经元有关的细胞特异性测定系统 项目＃1旨在开发与生理相关的离体模型，以筛选功能 选择性药物。同时，我们希望找到能在功能上反映多巴胺神经元的不朽细胞系。事先的 使用MN9D细胞系的工作表明，这些单元在功能上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，我们发现这就像是 不稳定，并寻求可以替换它的线。我们已经确定了两个出色的候选人出于我们的目的 （N27和CAD），我们首先将这些线表征适当的表型，并为其稳定性 当分子操纵时。