Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 9157486
• 负责人: David Sibley
• 金额: $ 172.82万
• 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/9157486
• 关键词: Adverse effects; Affect; Affinity; Agonist; Amino Acid Sequence; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Categories; Cell Culture Techniques; Cell Line; Cellular biology; Characteristics; Chemistry; DRD2 gene; Development; Disease; Docking; Dopamine; Dopamine Agonists; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Drug Kinetics; Drug Targeting; Etiology; Exhibits; Family; GTP-Binding Proteins; Goals; In Vitro; Inhibitory Concentration 50; Investigation; Lead; Libraries; Ligands; Mediating; Membrane; Mental disorders; Molecular; Molecular Bank; Molecular Models; Nature; Neurons; Neurotransmitter Receptor; Neurotransmitters; Nitrogen; Parents; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Property; Publishing; RBM5 gene; Receptor Signaling; Recruitment Activity; Regulation; Research; Signal Transduction; Solubility; Structural Models; Structure; Structure-Activity Relationship; System; Technology; Testing; Therapeutic; Therapeutic Agents; Triage; United States National Institutes of Health; alkalinity; analog; base; beta-arrestin; dopamine D3 receptor; dopamine D4 receptor; dopamine D5 receptor; high throughput screening; hydroxyl group; improved; in vivo; interest; molecular modeling; nervous system disorder; neuropsychiatry; neurorestoration; novel; pharmacophore; programs; radioligand; receptor; receptor coupling; receptor expression; response; scaffold; screening; small molecule; small molecule libraries; synthetic drug; tool; trafficking

There are five major types of dopamine receptors, classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, D2-like family consists of the D2, D3, and D4 dopamine receptors. The D2 receptor is important in the etiology and/or therapy of many neuropsychiatric diseases and is an FDA-validated drug target. Unfortunately, truly specific compounds for this receptor have been difficult to obtain due to the highly conserved amino acid sequences that form the orthosteric-binding site, where dopamine and other synthetic drugs bind to the receptor. As a result, drugs that target the D2 receptor frequently interact with other dopamine and non-dopamine receptors leading to unwanted side effects during disease treatment. In order to identify novel D2 receptor-selective compounds, a high throughput screen (HTS) of nearly 400,000 compounds in the NIH-MLPCN library was executed. This effort lead to the identification of a compound (MLS6916) with high affinity and selectivity for the D2 receptor. This compound was subjected to an iterative chemistry approach to improve its drug-like characteristics. Twenty-seven new analogs were synthesized with substituent changes on a separate fragment of the parent molecule. These analogs were studied using a beta-arrestin recruitment assay to determine functional IC50 values for a variety of D2-like receptors including D2, D3, and D4. The parent compound, MLS6916, displayed a 484-fold D2/D3 selectivity, however the compound exhibited only 24-fold D2/D4 selectivity. Of the newly synthesized compounds tested, MLS8039 showed the most promise, with a 1,000-fold D2/D3 selectivity and a 1,200-fold D2/D4 selectivity, indicating that it may be even more D2 selective than the parent scaffold. Further pharmacokinetic screening revealed that, while compound MLS8039 is not as metabolically stable as necessary for drug-like applications, it is very permeable with relatively good solubility. We have established important structure-activity relationships (SAR) around the parent compound and were able to produce more selective analogs of a D2 receptor antagonist. Further chemistry will be completed around the new lead compound MLS8039 with the aim to improve stability and solubility while maintaining its high selectivity. 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 finely-tuned study of receptor signaling. We previously published the discovery of a highly efficacious, functionally biased D2 dopamine receptor agonist (MLS1547) that can selectively activate G protein signaling while blocking beta-arrestin recruitment. In the interest of understanding the basis for its bias, the signaling properties of 23 MLS1547 analogs were characterized, which ranged from highly biased to unbiased. These results provided the basis for developing structure-activity relationships using pharmacophore modeling and molecular docking analyses. Subsequently, 69 additional MLS1547 analogs were tested for both beta-arrestin and G protein signaling, which refined our model of the structural basis for G protein bias. We have confirmed that a hydrophobic feature, like the chloro group at C5 of MLS1547 is required for G protein-biased signaling. A hydroxyl group para to the C5 group also correlates with strong biased signaling. Replacing the 2-pyridyl group with other nitrogen heterocycles alters the nitrogen basicity, and has a more nuanced effect on bias and signaling strength. Bicyclic scaffolds afforded more potent analogs than the monocyclic scaffolds investigated. Interestingly, some analogs switched from G protein to beta-arrestin bias, opening up additional, interesting avenues for understand the mechanism of biased D2R signaling. This, along with medicinal chemistry approaches will enable a highly targeted approach for investigating D2 receptor signaling in vivo, and may eventually broaden our understanding of pathological D2 receptor signaling in disease states. As an approach to discover highly selective allosteric modulators for the D3 dopamine receptor (DAR), our lab employed high throughput screening technologies. The NIH Molecular Libraries Program 400,000+ small molecule library was initially screened using a D3 DAR-mediated beta-arrestin recruitment assay. Confirmation and counter-screens were performed to obtain an initial assessment of selectivity and mechanisms of action and identified 57 potential negative allosteric modulators (NAMs), 63 potential positive allosteric modulators (PAMs), and 62 potential allosteric agonists. Further triage and characterization identified several D3 DAR-selective putative NAMs, PAMs, and allosteric agonists that are currently being characterized using additional assays to confirm their selectivity, activity, and mechanism of action. As D3-preferring D2/D3 DAR orthosteric agonists show promise as neuroprotective/neurorestorative agents, we conducted preliminary studies using one of the lead allosteric agonists, and found that it displays neuroprotective properties using a cell culture model system. We ultimately hope that these probes will prove useful as in vitro and in vivo pharmacological tools or leads for therapeutic drugs. 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 recently 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 thei

多巴胺受体有五种类型的类型，分为两种类似D1的主要类别，类似D2。 D1样系列由D1和D5多巴胺受体组成，而D2样系列由D2，D3和D4多巴胺受体组成。 D2受体在许多神经精神疾病的病因学和/或治疗中很重要，并且是FDA验证的药物靶标。不幸的是，由于高度保守的氨基酸序列形成了直角结合位点，多巴胺和其他合成药物与受体结合，因此很难获得该受体的真正特异性化合物。 结果，靶向D2受体的药物经常与其他多巴胺和非多巴胺受体相互作用，从而在疾病治疗过程中导致不必要的副作用。为了鉴定新型的D2受体选择性化合物，执行了NIH-MLPCN库中近400,000种化合物的高吞吐量屏幕（HTS）。这项工作导致鉴定具有高亲和力和对D2受体选择性的化合物（MLS6916）。 该化合物采用了一种迭代化学方法，以改善其类似药物的特性。 合成了27个新类似物，并在父分子的单独片段上与取代基变化。 使用Beta-arrestin募集测定法对这些类似物进行了研究，以确定多种D2样受体在内的功能性IC50值，包括D2，D3和D4。母体化合物MLS6916显示出484倍D2/D3的选择性，但是该化合物仅表现出24倍D2/D4的选择性。 在已测试的新合成化合物中，MLS8039显示出最大的希望，具有1,000倍D2/D3的选择性和1,200倍D2/D4的选择性，表明它可能比父母支架更具D2选择性。 进一步的药代动力学筛查表明，尽管化合物MLS8039在类似药物的应用中的代谢稳定不那么稳定，但相对较高的溶解度非常渗透。我们已经建立了围绕母体化合物周围的重要结构 - 活性关系（SAR），并能够产生D2受体拮抗剂的更有选择的类似物。将在新的铅化合物MLS8039周围完成进一步的化学，旨在提高稳定性和溶解度，同时保持其高选择性。 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精细调整的研究。 我们以前发表了发现高效，功能性的D2多巴胺受体激动剂（MLS1547），该发现可以选择性地激活G蛋白信号传导，同时阻止Beta-arrestin募集。为了理解其偏差的基础，表征了23 mLS1547类似物的信号传导特性，从高度偏见到无偏见。这些结果为使用药效团建模和分子对接分析建立结构活性关系提供了基础。随后，测试了69个MLS1547类似物的β-arrestin和G蛋白信号传导，这些模型完善了我们的G蛋白偏置结构基础模型。我们已经证实，G蛋白偏置信号传导需要一种疏水特征，例如MLS1547的C5的氯一组。与C5组的羟基PARA也与强有偏置的信号传导相关。用其他氮杂环代替2-吡啶基，会改变氮碱性，并对偏置和信号强度具有更细微的影响。与研究的单核脚手架相比，双环支架具有更强大的类似物。有趣的是，一些类似物从G蛋白转换为β-arrestin偏置，开辟了其他有趣的途径，以了解偏见的D2R信号传导的机制。与药物化学方法一起，这将实现一种高度针对性的方法来研究体内D2受体信号传导，并最终可能扩大我们对疾病状态中病理D2受体信号传导的理解。 作为发现D3多巴胺受体（DAR）高度选择性变构调节剂的一种方法，我们的实验室采用了高吞吐量筛选技术。 NIH分子库计划400,000以上的小分子库最初是使用D3 DAR介导的β-arrestin招聘测定法筛选的。进行确认和反屏幕，以获得对作用的选择性和机制的初步评估，并确定了57个潜在的负变构调节剂（NAM），63个潜在的阳性变构调节剂（PAM）和62个潜在的变构激动剂。进一步的分类和表征确定了几种D3 DAR选择性推定的NAM，PAM和变构激动剂，它们目前正在使用其他测定法来确认其选择性，活性和作用机理。由于D3优先的D2/D3 DAR正骨激动剂表现为神经保护性/神经遗传剂的希望，我们使用其中一项铅构层激动剂进行了初步研究，并发现它使用细胞培养模型系统显示出神经保护性质。我们最终希望这些探针将被证明是在体外和体内药理工具或治疗药物的铅。 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，我们最近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Thei