Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 7969514
• 负责人: David Sibley
• 金额: $ 135.9万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969514
• 关键词: ADRBK1 gene; Accounting; Adopted; Adverse effects; Agonist; Allosteric Site; Animal Model; Antiparkinson Agents; Antipsychotic Agents; Arrestins; Attenuated; Behavior; Binding; Biochemical; Biological Assay; Biological Availability; Bioluminescence; Cell Surface Receptors; Cell surface; Cellular biology; Characteristics; Complex; Coupling; Development; Disease; Dopamine; Dopamine D2 Receptor; Dopamine Receptor; Drug Delivery Systems; Dyes; Endocrine System Diseases; Endocytosis; Energy Transfer; Etiology; Exhibits; FDA approved; Family; Fluorescent Dyes; Future; G Protein-Coupled Receptor Signaling; G alpha q Protein; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Gilles de la Tourette syndrome; Goals; Hyperprolactinemia; In Vitro; Investigation; Ions; Lead; Ligand Binding; Ligands; Measures; Mediating; Membrane; Molecular; Molecular Bank; Molecular Cloning; Molecular Conformation; Mus; N-terminal; Neurology; Neurons; Neurotransmitter Receptor; Neurotransmitters; Parkinson Disease; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Phosphotransferases; Potassium; Property; Proteins; Psychiatry; Receptor Signaling; Regulation; Research; Restless Legs Syndrome; Role; Schizophrenia; Screening procedure; Signal Pathway; Signal Transduction; Small Interfering RNA; Specificity; Structure; System; Tardive Dyskinesia; Thallium; Therapeutic; Transducers; United States National Institutes of Health; Work; arrestin 2; base; drug discovery; efficacy testing; high throughput screening; improved; in vivo; interest; member; mutant; neuropsychiatry; novel; novel strategies; programs; receptor; receptor expression; repository; research study; small molecule; small molecule libraries; therapeutic target; tool; trafficking

In FY2009, we investigated the regulatory effects of GRK2 on D2 dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Over-expression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas siRNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D2 receptor as a phosphorylation-null receptor mutant was regulated similarly and over-expression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2, and several of its mutants, were able to co-immunoprecipitate with the D2 receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that over-expression of GRK2 attenuated the functional coupling of the D2 receptor and that this activity required the kinase activity of GRK2, but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2, but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates GPCR signaling in a manner independent of receptor phosphorylation. In FY2009, we continued working on our drug discovery project for allosteric ligands of the D2 receptor. G protein-coupled receptors (GPCRs) represent the largest family of therapeutic drug targets and account for the mechanisms of action of >60% of all FDA-approved drugs. Receptors for the neurotransmitter dopamine are members of this GPCR super-family and are involved in the etiology and/or therapy of a number of neuropsychiatric and endocrine disorders. In fact, amongst the dopamine receptors (DARs), the D2 subtype is arguably one of the most validated drug targets in neurology and psychiatry. For instance, all receptor-based antiparkinsonian drugs work via stimulating the D2 DAR whereas all FDA-approved antipsychotic agents are antagonists of this receptor. The D2 DAR is also therapeutically targeted in other disorders such as restless legs syndrome, tardive dyskinesia, Tourettes syndrome, and hyperprolactinemia. Most drugs targeting the D2 DAR are problematic, however, either being less efficacious as desired or possessing limiting side effects, most of which are due to cross-GPCR reactivity. One pharmacological approach towards improved target specificity is to identify allosteric ligands which bind to less conserved regions of receptors and therefore have the potential to be much more selective. Ligands that bind to such allosteric sites may promote conformation changes in the receptor that can produce positive or negative effects with respect to activation by the endogenous agonist, or in some cases can exhibit functional efficacy (agonist or inverse agonist) of their own. The goal of this project is to use high throughput screening (HTS) approaches to identify and develop novel small molecule allosteric modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. To this end, we propose to develop two assays capable of large-scale, high throughput screening of small molecule libraries. One assay involves the cellular co-expression of the D2 DAR with a chimeric Gq protein thus enabling the receptor to stimulate Ca2+ mobilization, which is detected through the activation of an intracellular fluorescent dye. The ssecond assay measures the ability of D2 DARs to promote the flux of thallium ion through G protein-regulated inward rectifying potassium (GIRK) channels, again measured through the activation of an intracellular dye. These assays will be configured into HTS formats and evaluated through the generation/calculation of Z parameters. The superior assay will be submitted for consideration by the Molecular Libraries Screening Centers Network (MLSCN) program for interrogation of the NIH Molecular Libraries small molecule repository. Secondary and counter-screening assays for other DAR subtypes will also be developed and implemented as necessary to confirm and validate MLSCN-generated hits. If needed, limited medicinal chemistry efforts will be performed to enhance the potency or efficacy, or the bioavailability of the most promising hit compounds. Future studies will be directed at evaluating the therapeutic potential of D2 DAR allosteric modulators using proof-of-concept efficacy tests in animal models of Parkinsons disease and schizophrenia. In FY2009, we also initiated a second drug discovery project related to improving therapeutics targeting the D2 receptor. A novel approach for attaining greater selectivity of therapeutic action is to identify and develop ligands that exhibit functionally selective properties. The phenomenon of functional selectivity, also termed biased agonism, protean agonism, agonist-directed trafficking, or collateral efficacy, is a relatively new concept in pharmacology and can occur when a receptor is able to transduce signals through more than one intracellular pathway. In this case, most agonists, in particular the endogenous transmitter will activate all signaling pathways in parallel with equal efficacy. However, it is now recognized, that some synthetic agonists may preferentially activate one pathway over another. While the mechanisms underlying functionally selective phenomena are not known with certainty, one hypothesis is that receptors can adopt multiple functionally active conformational states that are either stabilized or induced by selective ligands. In this scenario, some ligand-specific active conformations will selectively engage different G proteins or other signaling transducers such as β-arrestin. This may allow for the development of novel ligands that differentially activate (or block) a subset of signaling pathways for a single receptor, thus optimizing therapeutic action. The goal of this project is to use high throughput screening (HTS) approaches to identify and develop functionally-selective modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. We are particularly interested in developing probes that are functionally selective for the D2-DAR-stimulated β-arrestin signaling pathway, as previously all ligands for this receptor have been functionally characterized using G protein-mediated signaling. To this end, we will implement a high-fidelity bioluminescence resonance energy transfer (BRET)-based interaction assay for measuring agonist-induced recruitment of β-arrestin-2 to the D2 DAR. The assay will be configured into HTS format and submitted to the MLPCN program for interrogation of the NIH Molecular Libraries small molecule repository. Counter-screening assays will be developed and implemented to confirm and validate MLPCN-generated hits and to establish functionally selective characteristics of lead compounds. Such lead compounds should selectively stimulate, or block, D2 DAR/β-arrestin-mediated signaling in vivo, thus allowing for the of the role of this pathway in dopamine-regulated behaviors. Future studies will also evaluate the therapeutic potential of these functionally selective ligands via efficacy tests in animal models Parkinsons disease and schizophrenia.

2009财年，我们研究了GRK2对D2多巴胺受体信号传导的调节作用，发现该激酶以不依赖磷酸化的方式抑制受体表达和功能信号传导，显然是通过不同的机制。研究发现，GRK2 的过表达可抑制细胞表面的受体表达并增强激动剂诱导的内化，而内源性 GRK2 的 siRNA 敲低则导致细胞表面受体表达增加并减少激动剂介导的内吞作用。这些效应并不是由于 GRK2 介导的 D2 受体磷酸化所致，因为磷酸化无效受体突变体受到类似的调节，并且 GRK2 催化失活突变体的过度表达产生了相同的效应。受体表达的抑制与 GRK2 与受体复合物的组成型关联相关，因为我们发现 GRK2 及其几种突变体能够与 D2 受体共免疫沉淀。激动剂预处理并没有增强GRK2与受体共免疫沉淀的能力。 我们还发现GRK2的过度表达减弱了D2受体的功能偶联，并且这种活性需要GRK2的激酶活性，但不涉及受体磷酸化，因此表明额外的GRK2底物的参与。有趣的是，我们发现功能信号传导的抑制也需要GRK2的Gβγ结合活性，但不涉及GRK2 N端RH结构域。我们的结果提出了一种新机制，GRK2 通过该机制以不依赖于受体磷酸化的方式负向调节 GPCR 信号传导。 2009财年，我们继续致力于D2受体变构配体的药物发现项目。 G 蛋白偶联受体 (GPCR) 代表最大的治疗药物靶点家族，占 FDA 批准的所有药物的 60% 以上的作用机制。 神经递质多巴胺的受体是该 GPCR 超家族的成员，并参与许多神经精神和内分泌疾病的病因学和/或治疗。 事实上，在多巴胺受体 (DAR) 中，D2 亚型可以说是神经病学和精神病学中最有效的药物靶点之一。例如，所有基于受体的抗帕金森病药物都通过刺激 D2 DAR 发挥作用，而所有 FDA 批准的抗精神病药物都是该受体的拮抗剂。 D2 DAR 也是其他疾病的治疗靶点，例如不宁腿综合征、迟发性运动障碍、抽动秽语综合征和高催乳素血症。然而，大多数靶向 D2 DAR 的药物都存在问题，要么疗效不如预期，要么副作用有限，其中大部分是由于交叉 GPCR 反应性造成的。提高靶标特异性的一种药理学方法是鉴定与受体不太保守区域结合的变构配体，因此有可能更具选择性。与此类变构位点结合的配体可以促进受体的构象变化，从而对内源性激动剂的激活产生积极或消极的影响，或者在某些情况下可以表现出其自身的功能功效（激动剂或反向激动剂）。该项目的目标是使用高通量筛选 (HTS) 方法来识别和开发 D2 DAR 的新型小分子变构调节剂，用作体外和体内药理学工具以及动物模型的概念验证实验。神经精神疾病。 为此，我们建议开发两种能够大规模、高通量筛选小分子文库的测定方法。 一项检测涉及 D2 DAR 与嵌合 Gq 蛋白的细胞共表达，从而使受体能够刺激 Ca2+ 动员，这是通过细胞内荧光染料的激活来检测的。第二项测定测量了 D2 DAR 通过 G 蛋白调节的内向整流钾 (GIRK) 通道促进铊离子通量的能力，同样通过细胞内染料的激活进行测量。 这些测定将被配置为 HTS 格式，并通过 Z 参数的生成/计算进行评估。更好的检测将提交给分子图书馆筛选中心网络 (MLSCN) 计划考虑，以审讯 NIH 分子图书馆小分子存储库。 还将根据需要开发和实施其他 DAR 亚型的二次和反筛选测定，以确认和验证 MLSCN 生成的命中。如果需要，将进行有限的药物化学工作，以增强最有希望的热门化合物的效力或功效或生物利用度。 未来的研究将旨在通过帕金森病和精神分裂症动物模型的概念验证功效测试来评估 D2 DAR 变构调节剂的治疗潜力。 2009 财年，我们还启动了第二个与改进针对 D2 受体的疗法相关的药物发现项目。获得更大治疗作用选择性的新方法是识别和开发具有功能选择性特性的配体。功能选择性现象，也称为偏向激动、多变激动、激动剂定向运输或附带功效，是药理学中一个相对较新的概念，当受体能够通过多个细胞内途径转导信号时，就会发生这种现象。 在这种情况下，大多数激动剂，特别是内源性递质将以相同的功效并行激活所有信号传导途径。 然而，现在人们认识到，一些合成激动剂可能优先激活一种途径而不是另一种途径。虽然功能选择性现象背后的机制尚不清楚，但一种假设是受体可以采用多种功能活性构象状态，这些构象状态要么是稳定的，要么是由选择性配体诱导的。 在这种情况下，一些配体特异性活性构象将选择性地接合不同的 G 蛋白或其他信号传导转导物，例如 β-arrestin。这可能允许开发新的配体，差异性地激活（或阻断）单个受体的信号通路子集，从而优化治疗作用。 该项目的目标是使用高通量筛选 (HTS) 方法来识别和开发 D2 DAR 的功能选择性调节剂，用作体外和体内药理学工具以及神经精神动物模型的概念验证实验疾病。我们对开发对 D2-DAR 刺激的 β-arrestin 信号通路具有功能选择性的探针特别感兴趣，因为之前该受体的所有配体都已使用 G 蛋白介导的信号传导进行了功能表征。 为此，我们将实施基于高保真生物发光共振能量转移 (BRET) 的相互作用测定，用于测量激动剂诱导的 β-arrestin-2 向 D2 DAR 的招募。该检测将配置为 HTS 格式并提交给 MLPCN 计划，以供 NIH 分子图书馆小分子存储库查询。 将开发和实施反筛选测定法，以确认和验证 MLPCN 生成的命中并建立先导化合物的功能选择性特征。此类先导化合物应选择性地刺激或阻断体内 D2 DAR/β-抑制蛋白介导的信号传导，从而允许该途径在多巴胺调节行为中发挥作用。 未来的研究还将通过帕金森病和精神分裂症动物模型的功效测试来评估这些功能选择性配体的治疗潜力。