Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 8940034
• 负责人: David Sibley
• 金额: $ 175.89万
• 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/8940034
• 关键词: ADME Study; Adverse effects; Affect; Affinity; Agonist; Allosteric Site; Antipsychotic Agents; Behavioral; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cell Line; Cellular biology; Chemicals; Chinese Herbs; Classification; Cyclic AMP; Data; Development; Disease; Docking; Dopamine; Dopamine Agonists; Dopamine Antagonists; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Dopaminergic Agents; Drug Receptors; Etiology; Exhibits; Family; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; In Vitro; Individual; Investigation; Libraries; Ligands; Link; Mediating; Membrane; Mental disorders; Molecular; Molecular Bank; Molecular Models; Nature; Neurons; Neurotransmitter Receptor; Neurotransmitters; Pathologic Processes; Pathology; Pathway interactions; Pharmaceutical Chemistry; Physiology; Play; Property; Proteins; RBM5 gene; Receptor Activation; Receptor Signaling; Recruitment Activity; Regulation; Reporting; Research; Role; Signal Pathway; Signal Transduction; Structure; Structure-Activity Relationship; Symptoms; System; Testing; Therapeutic Agents; Triage; Uncertainty; United States National Institutes of Health; analog; base; beta-arrestin; dopamine D3 receptor; high throughput screening; in vivo; molecular modeling; nervous system disorder; neuropsychiatry; novel; pharmacophore; programs; radioligand; receptor; receptor coupling; receptor expression; research study; response; screening; serotonin receptor; small molecule; small molecule libraries; stepholidine; tool; trafficking

The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders and various D1R-selective ligands have shown potential as therapeutic agents. In an effort to identify novel selective allosteric modulators of the D1R we used a high throughput screening approach. 380,000 small molecules in the NIH Molecular Libraries Screening Center Network (MLPCN) library were screened using a cell line expressing the D1R coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature. We initially identified 96 putative positive allosteric modulator (PAM) hits that enhanced the EC20 activity of dopamine in the Ca2+ response and 6 of these were subsequently confirmed during triage and were chosen for further characterization. In addition we found approximately 115 agonist and 125 antagonist hits that failed to completely inhibit radioligand binding and thus have been classified as potential allosteric agonists and antagonists or negative allosteric modulators (NAMs). These compounds are currently being characterized using additional assays to confirm their selectivity, activity, and classification. Four of the PAM hits have selected for chemical optimization, which is ongoing now. The D2 dopamine receptor (D2R) is also involved in the etiology and/or therapy of many neuropsychiatric disorders. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within dopamine receptor (DAR) subtypes and other G protein-coupled receptors (GPCRs). We have employed a high throughput-screening approach using the 380,000 small molecule NIH MLPCN library to identify novel allosteric modulators of the D2R. The primary screen was conducted using a cell line expressing an inducible D2R coupled to a chimeric Gqi5 protein, thus linking receptor activation to Ca2+ mobilization. Hits were subjected to an extensive triage strategy to characterize DAR activity and selectivity. On the basis of these analyses, 745 agonist and 499 antagonist compounds were selected and evaluated using radioligand binding competition assays to identify the nature of their receptor interactions (orthosteric or allosteric). Compounds that are ineffective in competing for binding likely exert their functional activity via allosteric mechanisms. These experiments resulted in the identification of 47 agonists and 48 antagonists that had insignificant effects on radioligand binding when tested at concentrations up to 40 uM, despite exhibiting maximal functional effects at significantly lower concentrations. These compounds would thus appear to be allosteric agonists and negative allosteric modulators of the D2R. Further characterization is ongoing. The D2R can activate a spectrum of signaling cascades primarily through G proteins and beta-arrestin recruitment, making it an attractive target for the development of signaling biased ligands. Unlike dopamine, which simultaneously activates G proteins and recruits beta-arrestins, a biased ligand affects only one pathway, and the development of such ligands can allow for a more fine-tuned study of receptor signaling. Our lab has identified a compound (ML1547) that is a highly efficacious agonist at D2R-mediated G protein-linked signaling, but it does not recruit beta-arrestin. Rather, this compound is an antagonist of D2R-stimulated beta-arrestin-mediated signaling. A number of structural analogs of ML1547 were characterized for their signaling properties, which ranged from fully biased, partially biased, to unbiased. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally-selective properties of these compounds. This, along with medicinal chemistry approaches, will allow for the identification of more potent G protein-biased analogs of ML1547 that will enable a highly targeted approach for investigating D2R signaling in vivo, and may eventually allow for selective treatment of disease symptoms associated with D2R dysfunction. Many currently available dopaminergic drugs modulate both D2 and D3 DARs due to high homology in their orthosteric binding sites, leading to potential unwanted side effects, but also uncertainty as to the roles that each DAR subtype plays in normal and pathological processes. In order to discover compounds that target unique, less conserved allosteric sites of these DARs, our lab employed a high throughput screening approach. Through the NIH Molecular Libraries Program, compound 3843 was originally identified as a D2R antagonist in a screen of a 380,000+ small molecule library. Counter-screening assays of beta-arrestin recruitment revealed that this compound selectively activates the D3 DAR (D3R), yet also acts as an antagonist at the D2R. Over 270 analogs were synthesized and tested to explore the structure-activity relationship of 3843 for the D2R and D3R, and several compounds with a 1,000-fold or greater increase in D3R agonist potency were found. Initial competition binding assays suggest these compounds act in an allosteric manner at the D3R, and initial ADME studies performed on 3843 were favorable. We ultimately hope these probes will be useful as vitro and in vivo pharmacological tools to elucidate D3R specific physiology and pathology. The Chinese herbal extract (-)-stepholidine has been reported to exhibit D2R antagonism and D1R agonist-like activities in a number of in vivo behavioral analyses. On this basis, it has been proposed as a potential novel antipsychotic agent. Prior studies, however, have shown complicated and mixed pharmacological responses involving both dopamine and serotonin receptors. Importantly, clear in vitro characterization of (-)-stepholidnine's actions on individual DAR subtype signaling pathways has not been extensively explored. These data are particularly important given the recent discovery of the potential for signaling bias within the DAR family. In this study we investigated the biological actions and binding activities of (-)-stepholidine on all DAR subtypes. (-)-Stepholidine demonstrated relatively high binding affinity for all DAR subtypes, but failed to display any agonist response in either G protein- (cAMP assays and GTPgS binding), or beta-arrestin (beta-arrestin translocation assays)-mediated signaling at any DAR tested. Furthermore, (-)-stepholidine was a potent full antagonist of dopamine-mediated signaling of both D1- and D2-like receptors and did not display signaling bias, thereby demonstrating that it acts as a non-selective pan-dopamine receptor antagonist. Our conclusion is that (-)-stepholidine lacks agonist activity at D1Rs, as previously proposed, and does not exhibit functional selectivity for D2R signaling.

D1多巴胺受体（D1R）已与许多神经精神疾病有关，各种D1R选择配体显示出可能作为治疗剂的潜力。为了确定D1R的新型选择性变构调节剂，我们使用了高吞吐量筛选方法。 NIH分子文库筛选中心网络（MLPCN）库中的380,000个小分子使用表达与G15耦合的D1R的细胞系筛选，从而在受体刺激下产生了强大的Ca2+信号。通过次级功能和放射性置换结合测定测定，将HIT化合物分为三叶，以确定亚型选择性及其变构性与直角性质。 我们最初确定了96个假定的阳性变构调节剂（PAM）命中，从而增强了Ca2+反应中多巴胺的EC20活性，其中6个随后在分类过程中得到了证实，并选择了进一步的表征。 此外，我们发现大约有115个激动剂和125个拮抗剂命中未能完全抑制放射性结合，因此已被归类为潜在的变构激动剂和拮抗剂或负变构调节剂（NAM）。 这些化合物目前正在使用其他测定法来确认其选择性，活动和分类。其中四个PAM命中已选择进行化学优化，这正在进行中。 D2多巴胺受体（D2R）也参与了许多神经精神疾病的病因和/或治疗。不幸的是，该受体的真正特异性药物很难获得，这主要是由于多巴胺受体（DAR）亚型和其他G蛋白偶联受体（GPCR）中的正常结合位点的高保护。我们使用380,000个小分子NIH MLPCN库采用了高吞吐量方法来识别D2R的新型变构调节剂。 使用表达与嵌合GQI5蛋白的诱导D2R的细胞系进行了主要筛选，从而将受体激活与Ca2+动员联系起来。命中受到广泛的分类策略，以表征DAR活动和选择性。 根据这些分析，选择了745种激动剂和499种拮抗剂化合物并使用放射性结合竞争测定法进行评估，以识别其受体相互作用（正常或变构）的性质。 无效争夺结合的化合物可能通过变构机制发挥其功能活性。 这些实验导致鉴定了47种激动剂和48位拮抗剂，这些拮抗剂在浓度高达40 um时对放射性物体结合产生微不足道的作用，尽管在浓度明显较低的情况下表现出最大的功能作用。 因此，这些化合物似乎是D2R的变构激动剂和负变构调节剂。进一步的表征正在进行中。 D2R可以主要通过G蛋白和β-arrestin募集来激活信号级联的频谱，从而使其成为信号传导偏见配体的有吸引力的目标。与多巴胺同时激活G蛋白和募集β-art蛋白不同，有偏见的配体仅影响一条途径，并且这种配体的发展可以允许对受体信号传导进行更微观的研究。 我们的实验室已经确定了一种化合物（ML1547），该化合物是D2R介导的G蛋白连接信号的高效激动剂，但并未募集β-arrestin。 相反，该化合物是D2R刺激的β-arrestin介导的信号传导的拮抗剂。 ML1547的许多结构类似物的特征是其信号特性，这些特性从完全偏见，部分偏见到无偏见。这些结果为使用药效团建模和分子对接分析提供了基础，以建立这些化合物功能选择性特性的初步结构活性关系。这将与药物化学方法一起识别ML1547的更有效的G蛋白偏置类似物，该类似物将启用一种高度靶向的方法来研究体内D2R信号传导，并最终允许选择性治疗与D2R功能障碍相关的疾病症状。 许多目前可用的多巴胺能药物在其直角结合部位中的高同源性引起的D2和D3 DAR都会调节D2和D3 DAR，从而导致潜在的不良副作用，但对于每个DAR子类型在正常和病理过程中的作用而言也是不确定性。 为了发现针对这些DAR的独特，保守的变构位点的化合物，我们的实验室采用了高吞吐量筛选方法。 通过NIH分子库计划，在380,000多个小分子库的屏幕中，化合物3843最初被鉴定为D2R拮抗剂。 Beta-arrestin招募的反筛查测定法表明，该化合物选择性地激活了D3 DAR（D3R），但在D2R中也充当拮抗剂。 合成并测试了超过270个类似物，以探索D2R和D3R的3843的结构活性关系，并且发现了D3R激动剂效能的几种具有1,000倍或更高增加的化合物。 最初的竞争结合分析表明，这些化合物在D3R上以变构的方式作用，而对3843进行的初步ADME研究是有利的。 我们最终希望这些探针将作为体外和体内药理工具有用，以阐明D3R特定的生理和病理学。 据报道，在许多体内行为分析中，据报道，史蒂芬丁（Stepholidine）表现出D2R拮抗作用和D1R激动剂样活性。在此基础上，已提出它是一种潜在的新型抗精神病药。 然而，先前的研究表明，涉及多巴胺和5-羟色胺受体的复杂和混合的药理学反应。 重要的是，尚未广泛探索（ - ） - 斯蒂芬宁对单个DAR亚型信号途径的作用的清晰体外表征。 鉴于最近发现了DAR家族中信号偏差的潜力，这些数据尤其重要。 在这项研究中，我们调查了（ - ） - 斯蒂芬丁对所有DAR亚型的生物学作用和结合活性。 （ - ） - 斯蒂芬丁对所有DAR亚型表现出相对较高的结合亲和力，但未能在G蛋白（CAMP分析和GTPG结合）或Beta-arrestin（beta-arrestin易位分析）中显示任何激动剂反应。 此外，（ - ） - 斯蒂芬丁是D1-和D2样受体的多巴胺介导信号传导的有效完全拮抗剂，并且没有显示信号偏置，从而证明它充当非选择性泛胺受体受体拮抗剂。 我们的结论是（ - ）斯蒂芬丁（Stepholidine）如前所述，缺乏D1RS的激动剂活性，并且对D2R信号传导没有功能选择性。