Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 批准号: 10688924
• 负责人: David Sibley
• 金额: $ 291.75万
• 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/10688924
• 关键词: Affect; Affinity; Agonist; Allosteric Site; Animals; Antipsychotic Agents; Attenuated; Behavioral; Behavioral Assay; Binding; Binding Sites; Biochemical; Biological Assay; Bradykinesia; Brain; Catalepsy; Characteristics; Chemosensitization; Chimera organism; Chronic; Collection; Coupled; Delusions; Development; Disease; Docking; Dopamine; Dopamine Agonists; Dopamine Antagonists; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Dose; Drug Kinetics; Drug Receptors; Dyskinetic syndrome; Exhibits; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gambling; Goals; Gold; Half-Life; Hallucinations; Impaired cognition; Impairment; Impulse Control Disorders; Infusion procedures; Investigation; Kinetics; L-DOPA induced dyskinesia; Lead; Levodopa; Ligands; Locomotion; Mediating; Membrane; Mental disorders; Metabolic; Metabolism; Modality; Modeling; Modification; Molecular; Motivation; Motor; Mutagenesis; Mutation; Nerve Degeneration; Neurologic Process; Neurons; Neuroprotective Agents; Neurotransmitters; Oxidopamine; Parkinson Disease; Patients; Permeability; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Point Mutation; Property; Rattus; Receptor Signaling; Regulation; Research; Rodent; Role; Schizophrenia; Scoring Method; Signal Transduction; Site; Solubility; Structure-Activity Relationship; Symptoms; System; Testing; Therapeutic; Therapeutic Effect; Thiophenes; Time; Transmembrane Domain; Treadmill Tests; Tremor; Walking; Work; abnormal involuntary movement; analog; antagonist; beta-arrestin; clinical development; design; dopamine D3 receptor; dopaminergic neuron; drug development; experimental study; extracellular; high throughput screening; improved; in vivo; interest; iterative design; lead candidate; median forebrain bundle; nervous system disorder; neuropsychiatric disorder; novel; novel therapeutic intervention; novel therapeutics; oxidation; parkinsonian rodent; positive allosteric modulator; pramipexol; preference; receptor; receptor binding; receptor expression; ropinirole; scaffold; side effect; small molecule; trafficking

The D1 dopamine receptor (D1R) is a crucial regulator of dopaminergic signaling and is involved in neurological processes and diseases. 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 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 affinity for the D1R, although with different maximum efficacy and estimated Kb values. Combination experiments and receptor mutagenesis studies indicated that MLS1082 acts via the previously described intracellular loop-2 (ICL2) allosteric site targeted by two known D1R PAMs, Compound B and DETQ. MLS6585, however, does 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 the D2R, so loss of potentiation from the introduction of D2R sequence was used to detect potential regions of interest. This chimeric approach identified transmembrane region 7 (TM7) of D1R as a potential site for mediating MLS6585 activity. Further, specific point mutations identified residues near the extracellular region of TM7 that are required for MLS6585 PAM activity. These mutations had no effect on the activities of other PAMs binding to the ICL2 site. We used analog sets of MLS6585 to begin to understand structure-activity relationships underlying D1R allosteric modulation. In addition to validating the MLS6585 scaffold as a D1R PAM, the analogs implicate structural moieties that are crucial for PAM activity and receptor selectivity. Together, these efforts increase our understanding of D1R allosteric modulation as a means for developing novel therapeutic interventions. Schizophrenia is a devastating illness characterized by both positive (hallucinations, delusions) and negative (flat affect, decreased motivation) symptoms coupled with cognitive impairment. Current antipsychotic medications are effective in treating the positive symptoms through antagonism of the D2 dopamine receptor (D2R). However, antipsychotic treatment is also hindered by side-effects due to off-target activities at other GPCRs and unfavorable binding kinetics at the D2R. We have identified and characterized a novel D2R antagonist with high selectivity against other GPCRs ML321. In functional profiling screens of up to 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Schild-type functional assays revealed that ML321 acts as a competitive antagonist of the D2R while kinetic studies showed that ML321 exhibits slow-on and fast-off receptor binding rates; properties that are believed to limit extrapyramidal side-effects that are commonly observed with antipsychotics. In fact, using doses that were maximally effective in antipsychotic-predictive behavioral assays in rodents, ML321 promoted little to no catalepsy, suggesting that ML321 may produce less extrapyramidal side-effects in patients. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile supporting its development into an advanced drug lead. While a promising therapeutic, ML321 has a short metabolic half-life, impeding its clinical development. A metabolite study revealed that the primary site of metabolism involves oxidation of the alkyl-thiophene portion of ML321. To create more metabolically stable derivatives, we iteratively designed and synthesized over 100 analogs with modifications focused on the alkyl-thiophene moiety. These analogs were pharmacologically characterized for both D2R binding affinity and function, and were also tested for metabolic stability, permeability, and solubility. These efforts have led to the optimization of ML321 into a collection of lead candidates that show similar pharmacological characteristics of ML321, but with marked increases in metabolic stability and ADME properties. Molecular docking and mutagenesis studies have led to a better understanding of how ML321 binds to the D2R, which will further assist in analog design and development. Together, these findings have advanced our understanding of ML321 structure-activity relationships, particularly around the alkyl-thiophene moiety, and have identified lead candidates for in vivo pharmacokinetic studies, thus representing substantial progress in the development of a new antipsychotic treatment. Since its first use in treating Parkinsons disease (PD), L-DOPA has remained the gold standard of therapy for this disorder, defined by the progressive degeneration of dopaminergic neurons in the CNS leading to profound bradykinesia and tremor. The efficacy of L-DOPA wanes over time and is associated with increasing side effects, including motor fluctuations and dyskinesias. Several dopaminergic agonists have also been introduced to treat PD, including pramipexole and ropinirole, which exhibit fewer motor side effects but are associated with impulse control disorders such as excessive gambling and hypersexuality. Notably, the dopamine receptor subtype(s) mediating the therapeutic actions and/or side effects in PD therapy remain unknown. However, the preference of pramipexole and ropinirole for the D3 dopamine receptor (D3R) suggests the involvement of this subtype, although these drugs also activate the D2R at therapeutic doses. Importantly, no drug currently employed to treat PD alters the course of the disease and the discovery of neuroprotective agents remain an unmet need in PD therapeutics. Recently, we discovered a novel, potent and highly selective agonist for the D3R, ML417, that is brain penetrant and was found to protect against 6-OHDA-induced neurodegeneration of dopaminergic neurons (Moritz et al., J. Med. Chem. 63: 5526, 2020). In the current study, we used ML417 to probe the role of the D3R in a rat model of PD. We initially sought to investigate the role of the D3R in ameliorating bradykinesia in a hemi-parkinsonian rat model induced by 6-OHDA infusion into the medial forebrain bundle. Using a validated cylindrical treadmill test of locomotion, doses of ML417 up to 20 mg/kg had no effect on improving impairments in walking as assessed by step counts in the hemi-parkinsonian rats. In contrast, administration of L-DOPA (6 mg/kg) significantly improves locomotion in the same model. Further, pretreatment with a D3R-selective antagonist, SB277011A (30 mg/kg), did not attenuate the effects of L-DOPA in reducing bradykinesia. These results suggest that the D3R does not mediate the anti-bradykinetic effects of current PD therapeutics. However, we hypothesize that D3R stimulation may be beneficial for the treatment of L-DOPA-induced dyskinesias (LIDs) in PD. To test this, we used a chronic L-DOPA administration paradigm (12 mg/kg/day for 7 days) to induce dyskinesias in the hemi-parkinsonian rats. Subsequently, the effects of ML417 and SB277011A were assessed in these animals using an abnormal involuntary movement (AIMs) scoring method. Pretreatment with a single dose of ML417 (20 mg/kg) significantly reduced the intensity and duration of dyskinesias promoted with a single dose of L-DOPA (6 mg/kg). Further, co-administration of SB277011A (30 mg/kg) with ML417 attenuated the anti-dyskinetic effects of ML417, suggesting that the benefit is D3R-mediated. Overall, this study implies that D3R stimulation has no therapeutic effect on bradykinesia in PD, however, it may be beneficial in treating dyskinesias arising from L-DOPA therapy.

D1多巴胺受体（D1R）是多巴胺能信号传导的关键调节剂，参与神经过程和疾病。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都增强了激动剂刺激的G蛋白和β-arrestin介导的信号传导，并增加了D1R的多巴胺亲和力，尽管具有不同的最大功效和估计的KB值。组合实验和受体诱变研究表明，MLS1082通过先前描述的细胞内环-2（ICL2）变构位点起作用，该位点由两个已知的D1R PAM靶向，即化合物B和DETQ。但是，MLS6585不通过此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上没有PAM活性，因此使用D2R序列引入的增强损失被用于检测潜在的目标区域。这种嵌合方法将D1R的跨膜区域7（TM7）确定为介导MLS6585活性的潜在位点。此外，特定点突变确定了MLS6585 PAM活性所需的TM7细胞外区域附近的残基。这些突变对与ICL2位点结合的其他PAM的活性没有影响。我们使用了MLS6585的模拟集，以开始理解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，模拟还暗示了对PAM活性和受体选择性至关重要的结构部分。这些努力共同加剧了我们对D1R变构调节的理解，作为开发新型治疗干预措施的一种手段。 精神分裂症是一种毁灭性疾病，其特征是阳性（幻觉，妄想）和阴性（平坦影响，动机降低）症状以及认知障碍。当前的抗精神病药通过D2多巴胺受体（D2R）的拮抗作用可有效治疗阳性症状。然而，由于其他GPCR的脱靶活动以及D2R的不利结合动力学，抗精神病药物也受到副作用的阻碍。我们已经确定并表征了一种新型的D2R拮抗剂，具有高选择性对其他GPCR的ML321。在高达168种不同GPCR的功能分析筛选中，ML321在D2R的有效抑制之外表现出的活性很小，在较小程度上，D3R表现出异常的GPCR选择性。 Schild型功能分析表明，ML321充当D2R的竞争拮抗剂，而动力学研究表明ML321表现出缓慢的和快速的受体结合速率。据信可以限制抗精神病药通常观察到的特性。实际上，使用在啮齿动物中最大程度地有效的抗精神病药预测行为测定的剂量，ML321几乎没有促进抗衰病，这表明ML321在患者中可能会产生较少的锥体外副作用。重要的是，没有其他D2R拮抗剂表现出这种药理和行为概况，将其发育支持为先进的药物铅。 ML321是一种有前途的治疗性，具有短暂的代谢半衰期，阻碍了其临床发育。一项代谢物研究表明，代谢的主要部位涉及ML321的烷基噻吩部分的氧化。为了创建更代谢稳定的衍生物，我们在100多种类似物上进行了迭代设计和合成，其修饰集中在烷基 - 噻吩部分。这些类似物在药理学上均针对D2R结合亲和力和功能进行了药理表征，还测试了代谢稳定性，渗透性和溶解度。这些努力导致ML321的优化成一组铅候选者，这些候选者显示了ML321相似的药理特征，但代谢稳定性和ADME特性的增长显着增加。分子对接和诱变研究已经使ML321如何与D2R结合，这将进一步有助于模拟设计和开发。总之，这些发现使我们对ML321结构活性关系，尤其是在烷基噻吩部分中的一部分，并确定了体内药代动力学研究的主要候选者，从而代表了新的抗精神病药物的发展中的实质性进展。 自从首次用于治疗帕金森氏病（PD）以来，L-DOPA仍然是该疾病的金标准，这是由中枢神经系统中多巴胺能神经元的进行性变性所定义的，导致了深度的头屈肌和震颤。 L-DOPA随着时间的推移的疗效，与副作用的增加有关，包括运动波动和运动障碍。还引入了几种多巴胺能激动剂来治疗PD，包括pr曲甲烯和Ropinirole，它们表现出较少的运动副作用，但与脉冲控制障碍（例如过度赌博和性交性）有关。值得注意的是，介导PD治疗中介导的治疗作用和/或副作用的多巴胺受体亚型尚不清楚。然而，尽管这些药物在治疗剂量时也激活了D2R，但对D3多巴胺受体（D3R）的偏爱表明了这种亚型的参与。重要的是，目前尚无用来治疗PD的药物改变疾病的进程，而在PD治疗中发现神经保护剂仍然是未满足的需求。最近，我们发现了D3R，ML417的一种新颖，有效且高度选择性的激动剂，该激动剂是大脑渗透剂，被发现可以预防6-OHDA诱导的多巴胺能神经元的神经变性（Moritz等，J。Med。Chem。63：5526，2020）。在当前的研究中，我们使用ML417来探测D3R在PD大鼠模型中的作用。我们最初试图研究D3R在改善Bradykinesia中的作用，这是由6-OHDA输注到内侧​​前脑束引起的Hemi-Parkinsonian大鼠模型中的作用。使用经过验证的圆柱跑步机运动测试，高达20 mg/kg的ML417剂量对改善步行障碍的影响没有影响，如Hemi-Parkinsonian大鼠的步骤计数所评估。相反，L-DOPA（6 mg/kg）的给药可显着改善同一模型的运动。此外，使用D3R选择性拮抗剂SB277011A（30 mg/kg）进行预处理，并未衰减L-DOPA在减少Bradykinesia中的影响。这些结果表明，D3R不会介导当前PD疗法的抗胸运动作用。但是，我们假设D3R刺激可能有益于治疗PD中L-DOPA诱导的运动障碍（LIDS）。为了测试这一点，我们使用了慢性L-DOPA给药范例（12 mg/kg/day，持续7天）来诱导Hemi-Parkinsonian大鼠的运动障碍。随后，使用异常非自愿运动（AIMS）评分方法在这些动物中评估了ML417和SB277011a的作用。单剂量的ML417（20 mg/kg）预处理可显着降低用单剂量的L-DOPA（6 mg/kg）促进的运动障碍的强度和持续时间。此外，SB277011A（30 mg/kg）与ML417的共同给药减弱了ML417的抗肌气作用，这表明该益处是D3R介导的。总体而言，这项研究表明，D3R刺激对PD中的肌脱水没有治疗作用，但是，它可能有益于治疗由L-DOPA疗法引起的运动障碍。