Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 10263008
• 负责人: David Sibley
• 金额: $ 321.91万
• 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/10263008
• 关键词: Adult; Affinity; Agonist; Allosteric Site; Ames Assay; Amphetamines; Animal Disease Models; Antipsychotic Agents; Attenuated; Basal Ganglia; Behavioral; Behavioral Assay; Binding; Binding Sites; Biochemical; Biological Assay; Bradykinesia; Brain; Brain region; Caenorhabditis elegans; Catalepsy; Cells; Chemosensitization; Chimera organism; Clinical; Clinical Research; Cytometry; Development; Disease Progression; Dopamine; Dopamine Antagonists; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Dose; Drops; Drug Receptors; Drug Targeting; Exhibits; FDA approved; Fluorescence; Functional disorder; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gait abnormality; Genes; Genetic; Goals; Half-Life; Human; In Vitro; Investigation; Kinetics; LRRK2 gene; Lasers; Lead; Libraries; Ligands; Link; Measurement; Mediating; Membrane; Mental disorders; Metabolic; Modality; Modeling; Molecular; Molecular Bank; Monitor; Motor; Motor Activity; Mutagenesis; Nerve Degeneration; Neurodegenerative Disorders; Neurologic Process; Neurons; Neurotoxins; Neurotransmitters; Output; Parkinson Disease; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Plasma; Point Mutation; Property; Psychiatry; RNA Interference; Receptor Signaling; Regulation; Reporting; Reproducibility; Research; Rodent; Safety; Schizophrenia; Signal Transduction; Site; Solubility; Structure; Structure-Activity Relationship; Substance Use Disorder; Symptoms; Syndrome; System; Testing; Therapeutic; Time; Toxic effect; Tremor; United States National Institutes of Health; Work; alpha synuclein; analog; attenuation; beta-arrestin; brain dysfunction; cross reactivity; cytotoxicity; dopamine D3 receptor; dopaminergic neuron; drug candidate; drug craving; drug development; expectation; experimental study; extracellular; high throughput screening; in vivo; in vivo Model; interest; mutant; nervous system disorder; neuropsychiatric disorder; new therapeutic target; novel; novel therapeutics; positive allosteric modulator; predictive modeling; predictive test; prepulse inhibition; programs; progressive neurodegeneration; promoter; protein activation; radioligand; receptor; receptor expression; recruit; response; scaffold; screening; side effect; small molecule; small molecule libraries; targeted treatment; trafficking

The D1 dopamine receptor (D1R) is a crucial regulator of dopaminergic signaling and is involved in many neurological processes. It is an attractive target for treating neuropsychiatric disorders, however, the liabilities of orthosteric agonists have curtailed this treatment modality. Positive allosteric modulators (PAMs) of the D1R are therefore an alternative drug development strategy. We discovered two structurally distinct D1R PAMs via a high-throughput screen: MLS1082 and MLS6585. Both PAMs potentiate agonist-stimulated G-protein and beta-arrestin-mediated signaling and increase dopamine's affinity for the D1R. Combination experiments and receptor mutagenesis studies indicated that MLS1082 acts via the previously described ICL2 PAM binding site targeted by two known D1R PAMs, Compound B and DETQ. MLS6585 however did not act via this ICL2 site. To identify the MLS6585 binding site, chimeras of the D1R and D2R were used. MLS6585 has no PAM activity at D2R, so loss of potentiation was used to detect potential regions of interest. This chimeric approach identified TM7 of the D1R as a potential region for mediating MLS6585 activity. Further, specific point mutations identified residues near the extracellular tip of TM7 that are required for the PAM activity of MLS6585 with no effect on MLS1082 activity. We used analog sets of MLS6585 to begin to understand the structure-activity relationships underlying D1R allosteric modulation. In addition to validating the MLS6585 scaffold as a D1R PAM, the analogs suggest structural moieties crucial for PAM activity and selectivity. Together, these efforts increase our understanding of D1R allosteric modulation for development of potential therapeutics. The D2 dopamine receptor (D2R) is one of the most validated drug targets in psychiatry and there is a strong correlation between the clinical doses of antipsychotics and their potencies for blocking D2Rs. However, current FDA-approved antipsychotic drugs typically cross-react with other GPCRs, leading to many deleterious side-effects. We have now identified and characterized a lead D2R antagonist with high GPCR selectivity, ML321. ML321 shows little GPCR cross-reactivity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Behavioral assays in rodents demonstrate ML321's efficacy in models that are predictive of antipsychotic effects in humans (e.g., attenuation of both amphetamine and PCP-induced locomotor activity and pre-pulse inhibition). Importantly, using doses that are maximally effective in antipsychotic-predictive assays, ML321 promotes little to no catalepsy, suggesting that ML321 may produce fewer extrapyramidal side effects in patients (an on-target side effect). This property may be due to unique binding kinetics of ML321 at the D2R. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile, which supports ML321's promise as a superior therapeutic. No concerns were noted during safety/toxicity studies including hERG channel activation, cytotoxicity, an AMES test, and a CYP inhibition study. ML321 demonstrates good brain penetrability (20% vs. plasma), but also exhibits a relatively short half-life in vivo of about 2 hr. The latter property may represent an impediment to the advancement of this antipsychotic candidate into clinical studies. Approximately 50 ML321 analogs have been synthesized and are being characterized pharmacologically using D2R radioligand binding and functional assays. In parallel, these structural analogs are being assessed in vitro for ADME in order to determine their metabolic stability, solubility and predicted penetrability. Overall, our expectation is that the ML321 scaffold can be optimized into an advanced drug candidate with the potential for targeting schizophrenia and other psychotic syndromes involving the D2R. Due to the limited distribution of the D3 dopamine receptor (D3R) in limbic regions of the brain, D3R-selective antagonists may be useful as therapeutics for substance use disorders (SUDs) as they could attenuate drug craving symptoms without the motor side effects frequently incurred by D2R-preferring antagonists. To find highly selective allosteric antagonists of the D3R, our lab utilized a high-throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library using a D3R-mediated beta-arrestin recruitment assay. We found one compound, MLS6357, that was selective for the D3R versus the D2R in several functional outputs including beta-arrestin recruitment and G-protein activation. Further, radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross-reactivity with other GPCRs. Additionally, Schild-type functional assays found that MLS6357 is a purely non-competitive negative allosteric modulator of the D3R. We synthesized 46 analogs of MSL6357 using iterative medicinal chemistry and tested them for activity which revealed structure activity relationships and further refinement of the scaffold. These efforts produced modulators that are 5- to 9-fold more potent than the original hit compound. To identify the allosteric site on the D3R we utilized D3R/D2R chimeras which revealed receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R-related pathophysiology. Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non-motor complications. There are currently no drugs to halt the progression of this disease. Although loss of dopamine in the basal ganglia is recognized as the hallmark of PD, the molecular mechanisms underlying this loss and subsequent brain dysfunction remain poorly characterized. The majority of PD animal models involve the administration of neurotoxins that target dopaminergic neurons leading to their degeneration, however these models correlate poorly with the disease progression in humans. More accurate models may utilize genetic modulation of PD-related genes and exhibit progressive neurodegeneration. Here, we report the development of a high-throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans). Two strains of C. elegans containing human PD-linked genes were used, one expressing mutant (A53T) alpha-synuclein, and the other expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2). Both strains express GFP in their dopaminergic neurons and the lines were further crossed into a neuronal RNAi-sensitive background strain expressing mCherry under a pharyngeal promoter. Daily measurements of GFP/mCherry fluorescence intensity were performed using laser cytometry of worms sorted into 384-well microplates. Robust temporal degeneration of dopaminergic neurons was found to occur within the first eight days of adulthood in the C. elegans models of PD, but not in a wild-type control strain. The LRRK2 model was particularly severe as total GFP intensity dropped by 75-85% during the time of assay. We determined a signal (cell loss) to baseline ratio of approximately 4-fold, sufficient for screening applications. Our results indicate that this assay provides a reproducible high-throughput measurement of dopaminergic neurodegeneration using an in vivo model. Future studies may exploit this model to conduct quantitative high-throughput screens to identify small molecules capable of inhibiting this neurodegeneration or to use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for the treatment of PD.

D1多巴胺受体（D1R）是多巴胺能信号传导的关键调节剂，参与了许多神经系统过程。它是治疗神经精神疾病的有吸引力的靶标，但是，正常激动剂的责任减少了这种治疗方式。因此，D1R的阳性变构调节剂（PAM）是一种替代性药物开发策略。我们通过高通量屏幕发现了两个结构上不同的D1R PAM：MLS1082和MLS6585。两种PAMS增强了激动剂刺激的G蛋白和β-arrest蛋白介导的信号传导，并增加了多巴胺对D1R的亲和力。组合实验和受体诱变研究表明，MLS1082通过先前描述的ICL2 PAM结合位点起作用，这些ICL2 PAM结合位点由两个已知的D1R PAMS，即化合物B和DETQ。但是，MLS6585未通过此ICL2站点起作用。为了识别MLS6585结合位点，使用了D1R和D2R的嵌合体。 MLS6585在D2R时没有PAM活性，因此使用增强损失来检测潜在的目标区域。这种嵌合方法将D1R的TM7确定为介导MLS6585活性的潜在区域。此外，特定点突变确定了MLS6585的PAM活性所需的细胞外尖端附近的残基，对MLS1082活性没有影响。我们使用MLS6585的模拟集来理解D1R变构调制基础的结构活动关系。除了将MLS6585支架验证为D1R PAM外，模拟表明结构部分对于PAM活性和选择性至关重要。这些努力共同加剧了我们对D1R变构调节的理解，以开发潜在的治疗剂。 D2多巴胺受体（D2R）是精神病学中最有效的药物靶标之一，抗精神病药的临床剂量与阻断D2RS的效力之间存在很强的相关性。但是，当前FDA批准的抗精神病药通常与其他GPCR进行交叉反应，从而导致许多有害的副作用。现在，我们已经确定并表征了具有高GPCR选择性ML321的铅D2R拮抗剂。 ML321几乎没有GPCR交叉反应性超出D2R的抑制，并且在较小程度上表现出了异常的GPCR选择性。啮齿动物的行为测定表明，ML321在预测人类抗精神病药作用的模型中的功效（例如，苯丙胺和PCP诱导的运动活性和脉冲抑制作用）。重要的是，使用在抗精神病药预测性测定中具有最大有效性的剂量，ML321几乎没有促进培养基，这表明ML321在患者中可能会产生较少的锥体外副作用（靶标副作用）。 该特性可能是由于D2R上ML321的独特结合动力学引起的。重要的是，没有其他D2R拮抗剂表现出这种药理和行为特征，它支持ML321作为优质治疗的承诺。在安全/毒性研究中，没有注意到包括HERG通道激活，细胞毒性，AMES检测和CYP抑制研究的问题。 ML321表现出良好的大脑渗透性（20％与等离子体），但在体内的半衰期相对较短，约为2小时。后一种特性可能代表了该抗精神病药候选者在临床研究中发展的障碍。大约50个ML321类似物已经合成，并使用D2R放射性结合和功能测定法对药理学进行了表征。同时，这些结构类似物正在体外评估以确定其代谢稳定性，溶解度和预测的渗透性。 总体而言，我们的期望是，可以将ML321支架优化为靶向精神分裂症和其他涉及D2R的精神分裂综合症的潜力。 由于D3多巴胺受体（D3R）在大脑的边缘区域的分布有限，因此D3R选择性拮抗剂可能是有用的，可以作为药物使用障碍（SUD）的治疗剂，因为它们可以减轻不受D2R prefrigry prefrigrigry拮抗剂的运动副作用而使药物渴望的症状。为了找到D3R高度选择性的变构拮抗剂，我们的实验室利用了NIH分子库计划的高通量屏幕，使用D3R介导的β-arrestin募集分析方法400,000以上的小分子库。我们发现了一种化合物MLS6357，它在多个功能输出中与D2R相比，在包括β-arrestin募集和G蛋白激活的几个功能输出中具有选择性。此外，使用密切相关的GPCR的放射性配体结合和功能分析表明，MLS6357与其他GPCR的交叉反应非常有限。 此外，Schild型功能测定法发现MLS6357是D3R的纯粹非竞争性负变构调节剂。我们使用迭代药物化学合成了46个MSL6357的类似物，并测试了它们的活性，这揭示了结构活动关系并进一步完善支架。 这些努力产生的调节剂比原始命中化合物高5至9倍。为了识别D3R上的变构位点，我们使用了D3R/D2R嵌合体，该嵌合体揭示了复合疗效所需的受体区域。 MLS6357的结合口袋的进一步细化将为未来的药物化学工作提供信息。最终，这种新颖的支架可能是D3R相关病理生理学的药理探针或治疗铅。 帕金森氏病（PD）是一种神经退行性疾病，其特征是多巴胺能神经元的丧失，导致胸肌疾病，震颤，步态异常和许多非运动并发症。目前没有药物可以阻止这种疾病的进展。尽管基底神经节中多巴胺的丧失被认为是PD的标志，但这种损失和随后的脑功能障碍的分子机制仍然很差。大多数PD动物模型涉及靶向多巴胺能神经元导致其变性的神经毒素的施用，但是这些模型与人类疾病进展较差。更准确的模型可以利用PD相关基因的遗传调节并表现出进行性神经变性。在这里，我们报告了用于监测秀丽隐杆线虫（秀丽隐杆线虫）中多巴胺能神经变性的高通量测定法。使用了两种含有人类PD连接基因的秀丽隐杆线虫菌株，一种表达突变体（A53T）α-核蛋白，另一个表达突变体（G2019S）含亮氨酸富含亮氨酸的重复激酶2（LRRRK2）。两种菌株在其多巴胺能神经元中表达GFP，并将线进一步跨入咽部启动子下表达麦克利的神经元RNAi敏感背景菌株。使用蠕虫的激光细胞仪对GFP/MCHERRY荧光强度进行了每日测量，分为384孔微孔板。 在PD的秀丽隐杆线虫模型中，发现多巴胺能神经元的稳健时间变性发生，但在野生型对照菌株中不发生。 LRRK2模型特别严重，因为在测定期间，总GFP强度下降了75-85％。 我们确定了大约4倍的信号（细胞损失）与基线比率，足以筛查应用。我们的结果表明，该测定法提供了使用体内模型对多巴胺能神经变性的可重复性高通量测量。未来的研究可能会利用该模型进行定量的高通量筛选，以鉴定能够抑制这种神经变性的小分子或使用RNAi文库来鉴定介导神经退行性反应的基因，从而鉴定出用于PD处理的新药物。