Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 8556998
• 负责人: David Sibley
• 金额: $ 206.57万
• 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/8556998
• 关键词: Adverse effects; Affinity; Agonist; Antibodies; Antiparkinson Agents; Antipsychotic Agents; Arrestins; Behavior; Benzazepines; Binding; Binding Sites; Biochemical; Biological Assay; Cell Line; Cells; Cellular biology; Chemicals; Cherry - dietary; Clinical; Cyclic AMP; Data; Development; Disease; Dopamine; Dopamine Antagonists; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Drug Receptors; Endocytosis; Etiology; Exhibits; FDA approved; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; In Vitro; Individual; Investigation; Libraries; Life; Ligands; Link; Measurement; Measures; Mediating; Membrane; Membrane Potentials; Mental disorders; Molecular; Neurons; Neurotransmitter Receptor; Neurotransmitters; Pathway interactions; Production; Property; Proteins; Receptor Activation; Receptor Signaling; Regulation; Reporting; Research; Rodent; SKF38393; Screening Result; Screening procedure; Series; Signal Pathway; Signal Transduction; Structure; System; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Triage; Variant; analog; base; beta-Galactosidase; beta-arrestin; cyclic-nucleotide gated ion channels; fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether; high throughput screening; in vivo; nervous system disorder; neuropsychiatry; novel; radioligand; receptor; receptor coupling; receptor expression; receptor internalization; response; small molecule libraries; trafficking

The D2 dopamine receptor (DAR) is central in the etiology and/or therapy of many neuropsychiatric disorders. Specifically, D2 DAR antagonism is the hallmark of all FDA-approved antipsychotics and stimulation of the D2 DAR is critical for effective antiparkinsonian therapy. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within DAR subtypes and between other G protein-coupled receptors. We have developed a high throughput-screening (HTS) platform to interrogate large chemical compound libraries and screened a 370,000+ small molecule library was screened to identify agonists, positive allosteric modulators, or antagonists. The primary HTS assay utilizes a cell line expressing the D2 DAR coupled to a chimeric Gqi5 protein, thereby linking receptor activation to robust Ca2+ mobilization that is measured using a fluorescent readout. We have also developed HTS-formatted secondary assays to measure orthogonal D2 DAR activities (cAMP modulation, beta-arrestin interactions, GIRK channel activation) as well as counter-screening assays to determine selectivity between other DAR subtypes (D1, D3, D4 and D5). Interaction of hit ligands with the orthosteric binding site of the D2 DAR was assessed using standard radioligand binding competition assays. For Hit Compound A, analogs were made to deduce SAR and develop higher affinity analogs. The primary HTS-screen resulted in the identification of 2,288 compounds with agonist activity and 2,294 compounds with antagonist activity. These compounds were cherry-picked to maximize the chances of identifying chemical series with unique activities. Hits were subjected to orthogonal and counter-screening functional assays. While the primary goal for this screen was to identify allosteric compounds, a by-product of this screen and subsequent triaging was the identification of compounds that, while orthosteric, exhibit high selectivity for the D2 DAR and/or are functionally selective with respect to D2 DAR signaling pathways. One such chemotype discovered this way (compound A) selectively activates the D2 DAR in comparison with other DAR subtypes. We found that compound A exhibits full agonist activity with EC50 values ranging from 100 nM to 1 microM using three different functional assays for the D2 DAR; Ca2+ mobilization, inhibition of cAMP accumulation, and beta-arrestin recruitment. Using beta-arrestin recruitment assays to compare with other DARs, we found that compound A has no activity at D1-like DARs (D1 and D5) or on D4 DARs. However, compound A displays either weak partial agonist (<20% of the DA response) or full antagonist activity at D3 DARs. Radioligand binding assays revealed that compound A exhibits Ki values of 1 microM and 100 nM for the D2 and D3 DARs, respectively. Interestingly compound A is a full antagonist with no agonist activity on D2 linked GIRK channel activation. Furthermore, two closely related synthesized analogs of compound A show full D2 agonist activity in G-protein linked D2 functional assays and full antagonist activity in D3 assays (with 10% partial agonism). In summary, compound A is a full and selective agonist at G-protein-linked and arrestin mediated D2 DAR assays, however it shows antagonist activity in D2 GIRK channel assays. Furthermore it also functions as a D3 DAR antagonist on all D3 functional assays tested. This is the first known compound that functionally discriminates between the D2 and D3 DARs and selectively activates the D2 DAR in the absence of D3 DAR activation. The D1 dopamine receptor (DAR) has been implicated in numerous neuropsychiatric disorders, and various D1 ligands have shown great potential as therapeutic agents. However, numerous side effects have limited their clinical utility and it is unknown which D1-mediated signaling pathways are responsible for therapeutic effects versus side effects. Recently it has been appreciated that compounds exist that can modulate one signaling pathway while having no effect on other signaling pathways that are associated with the same receptor. These functionally selective compounds present a unique therapeutic opportunity to target individual pathways while minimizing signaling through others. Previous studies have identified substituted benzazepine compounds, including SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 as D1 agonists. However, the in vivo effects of these compounds have not correlated well to their in vitro pharmacological activities. This is especially true of SKF83959 that has been described as an atypical D1 agonist that does not induce the same behaviors in rodents as typical D1 agonists. A series of substituted benzazepines, and structurally dissimilar D1 agonists, were tested for their functional effects on D1-mediated cAMP accumulation, D1-mediated beta-arrestin recruitment, and D1 receptor internalization using live cell functional assays. With respect to beta-arrestin recruitment, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 were antagonists of this response whereas other substituted benzazepines were nearly full agonists with efficacies ranging from 60-75%. We also examined D1 receptor internalization using a novel beta-galactosidase complementation assay. As with the beta-arrestin recruitment assay, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 exhibited little agonist efficacy, yet the other substituted benzazepines were full agonists in terms of inducing receptor internalization. Our study also used two different D1 cAMP assays: DiscoveRx (DRX) HitHunter, an antibody-based assay that provides a direct measurement of cAMP accumulation, and Codex, which utilizes a cyclic-nucleotide gated channel to measure changes in membrane potential as an indirect readout of cAMP production. In the Codex assay, all compounds elicited an increase in D1 cAMP accumulation similar to that produced by dopamine, with efficacies ranging from 90-110% for the typical agonists and 50-100%for the atypical agonists, effects that were inhibited by the D1 antagonist SCH23390. In contrast, in the DRX D1 HitHunter cAMP assay, the typical agonists displayed a greater variation in agonist efficacy while the substituted benzazepines exhibited a range of efficacies similar to those seen in the Codex assay. Thus, we have identified a group of substituted benzazepine ligands, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 that are antagonists of D1 receptor-mediated recruitment of beta-arrestin and are devoid of agonist-induced receptor endocytosis. However, in contrast to previous reports, the atypical substituted benzazepines did show full-to-partial agonist activities in two assays of D1-mediated cAMP accumulation. Taken together, these data identify a novel group of substituted benzazepines that are functionally selective for the cAMP-mediated signaling pathway of the D1 receptor. These data may be useful in interpretation of many seemingly contrasting in vivo/in vitro effects of these compounds, and may be useful in further identification of pathway-selective ligands of the D1 dopamine receptor.

D2多巴胺受体（DAR）是许多神经精神疾病的病因和/或治疗中的核心。具体而言，D2 DAR拮抗作用是所有FDA批准的抗精神病药的标志，而D2 DAR的刺激对于有效的反帕金森疗法至关重要。 不幸的是，该受体的真正特异性药物很难获得，这主要是由于DAR亚型内部和其他G蛋白偶联受体之间的直角结合位点的高保守性。 我们已经开发了一个高通量筛选（HTS）平台来询问大型化学复合文库，并筛选了一个370,000多个小分子库，以识别激动剂，正变态调节剂或拮抗剂。 主要的HTS测定法使用了表达D2 DAR耦合到嵌合GQI5蛋白的细胞系，从而将受体激活与使用荧光读数测量的可靠Ca2+动员联系起来。 我们还开发了HTS形式的次级测定，以测量正交D2 DAR活动（CAMP调制，β-arrestin相互作用，Girk通道激活）以及反筛查测定法，以确定其他DAR子类型之间的选择性（D1，D3，D3，D4和D5）。 使用标准的放射线结合竞争测定法评估了HIT配体与D2 DAR的正常结合位点的相互作用。 对于命中化合物A，进行了类似物来推断SAR并产生较高的亲和力类似物。主要的HTS屏幕导致了2,288种具有激动剂活性的化合物和2,294种具有拮抗剂活性的化合物。 挑选了这些化合物，以最大程度地利用独特的活动识别化学系列的机会。 命中受到正交和反筛查功能测定。 虽然该屏幕的主要目标是识别变构化合物，但该屏幕的副产品和随后的三角剖分是鉴定化合物，而牙本质对D2 DAR和/或相对于D2 DAR信号途径具有较高的D2 DAR和/或在功能上具有选择性。 一种这样的化学型以这种方式发现（化合物A）与其他DAR亚型相比，有选择地激活D2 DAR。 我们发现，使用D2 DAR的三个不同功能测定法，复合A具有EC50值的全部激动剂活性。 CA2+动员，抑制营地积累和β-Arrestin招募。使用Beta-arrestin招聘测定法与其他DARS进行比较，我们发现复合A在D1样DARS（D1和D5）或D4 DARS上没有活动。 但是，化合物A显示出弱的部分激动剂（占DA响应的20％）或D3 DARS的完全拮抗剂活性。 放射性结合测定法显示，D2和D3 DAR的化合物A分别显示为1 microM和100 nm的Ki值。 有趣的是，化合物A是一个完整的拮抗剂，在D2连接的Girk通道激活上没有激动剂活性。 此外，化合物的两个密切相关的合成类似物在G蛋白链接的D2功能测定中显示了完全D2激动剂活性，并且在D3测定中（有10％的部分激动剂）中显示了完全的D2功能测定和完整的拮抗剂活性。 总而言之，化合物A是G蛋白连接和抑制蛋白介导的D2 DAR分析的完整而有选择性的激动剂，但是它显示了D2 Girk频道测定中的拮抗剂活性。 此外，它在所有测试的D3功能测定中也充当D3 DAR拮抗剂。这是第一个在功能区分D2和D3 DAR的已知化合物，并在没有D3 DAR激活的情况下选择性地激活D2 DAR。 D1多巴胺受体（DAR）与许多神经精神疾病有关，各种D1配体作为治疗剂的潜力很大。然而，许多副作用限制了其临床实用性，尚不清楚哪种D1介导的信号通路负责治疗效应与副作用。 最近，人们对存在可以调节一个信号通路的化合物，而对与同一受体相关的其他信号通路没有影响。 这些功能选择性化合物为靶向单个途径的独特机会提供了独特的治疗机会，同时通过其他人的信号最小化。先前的研究已经确定了替代的苯唑化化合物，包括SKF83959，SKF38393，SKF82957，SKF77434和SKF75670是D1激动剂。但是，这些化合物的体内作用与它们的体外药理活性没有很好的相关性。 SKF83959尤其如此，该SKF83959被描述为非典型D1激动剂，它不会在啮齿动物中与典型的D1激动剂相同的行为。测试了一系列取代的二苯甲酰苯并e和结构上不同的D1激动剂，该功能对D1介导的cAMP积累，D1介导的β-arrestin募集以及D1受体内部化的功能作用。关于Beta-Arrestin招聘，SKF83959，SKF38393，SKF82957，SKF77434和SKF77434和SKF75670是这种反应的拮抗剂，而其他替代的苯甲酰苯并e则几乎是60-75％的疗效。 我们还使用新型的β-半乳糖苷酶互补测定法检查了D1受体内在化。 与Beta-Arrestin招聘测定法一样，SKF83959，SKF38393，SKF82957，SKF77434和SKF75670几乎没有激动剂的功效，但其他替代的苯并e则是诱导受体内在化的全部激动剂。我们的研究还使用了两种不同的D1 CAMP分析：Discovex（DRX）Hithunter，一种基于抗体的测定法，可直接测量CAMP的积累，而Codex则利用环核苷酸门控通道来衡量膜潜能的变化，作为间接的营地产量读数。在法典测定中，所有化合物都引起了与多巴胺相似的D1 CAMP积累的增加，典型激动剂的效力从90-110％，非典型激动剂的50-100％不等，而D1 Antomanist抑制了D1 Anteragist Sch23390。相比之下，在DRX D1 Hithunter CAMP分析中，典型的激动剂表现出更大的激动剂功效变化，而取代的苯甲酰苯并epines则表现出与法典测定法相似的一系列疗效。 因此，我们已经确定了一组取代的苯甲酰苯甲皮配体，SKF83959，SKF38393，SKF82957，SKF77434和SKF75670，它们是D1受体受体介导的β-arta- arta- arta- rart蛋白的拮抗剂，并属于Agonists诱导的受体互联网。 但是，与以前的报道相反，非典型替代的苯甲酰苯甲苯甲烷确实在两种D1介导的营地积累的测定中表现出了全部至分子的激动剂活动。综上所述，这些数据确定了一组新型的取代苯并甲曲，对于D1受体的CAMP介导的信号通路具有功能性选择性。这些数据可能有助于解释这些化合物的许多看似对比的体内/体外作用，并且可能有助于进一步识别D1多巴胺受体的途径选择性配体。