Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

基本信息

项目摘要

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重要调节因子,参与神经系统过程和疾病。它是治疗神经精神疾病的一个有吸引力的靶点,然而,正位激动剂的缺点限制了这种治疗方式。因此,D1R 的正变构调节剂 (PAM) 是一种替代药物开发策略。我们通过高通量筛选发现了两种结构不同的 D1R PAM:MLS1082 和 MLS6585。两种 PAM 均可增强激动剂刺激的 G 蛋白和 β-抑制蛋白介导的信号传导,并增加多巴胺对 D1R 的亲和力,尽管最大功效和估计 Kb 值不同。组合实验和受体诱变研究表明,MLS1082 通过之前描述的细胞内 Loop-2 (ICL2) 变构位点发挥作用,该位点由两种已知的 D1R PAM(化合物 B 和 DETQ)靶向。然而,MLS6585 不通过该 ICL2 站点起作用。为了鉴定 MLS6585 结合位点,使用了 D1R 和 D2R 的嵌合体。 MLS6585 在 D2R 处没有 PAM 活性,因此引入 D2R 序列导致的增强作用损失可用于检测潜在的感兴趣区域。这种嵌合方法将 D1R 的跨膜区 7 (TM7) 鉴定为介导 MLS6585 活性的潜在位点。此外,特定的点突变鉴定了 TM7 胞外区域附近的残基,这些残基是 MLS6585 PAM 活性所需的。这些突变对其他 PAM 与 ICL2 位点结合的活性没有影响。我们使用 MLS6585 的模拟集来开始了解 D1R 变构调制背后的结构-活性关系。除了验证 MLS6585 支架作为 D1R PAM 之外,类似物还暗示了对 PAM 活性和受体选择性至关重要的结构部分。总之,这些努力加深了我们对 D1R 变构调节作为开发新型治疗干预手段的理解。 精神分裂症是一种毁灭性的疾病,其特征是积极的(幻觉、妄想)和消极的(情绪平淡、动机下降)症状以及认知障碍。目前的抗精神病药物通过拮抗 D2 多巴胺受体 (D2R) 来有效治疗阳性症状。然而,由于其他 GPCR 的脱靶活性和 D2R 的不利结合动力学,抗精神病治疗也受到副作用的阻碍。我们已经鉴定并表征了一种新型 D2R 拮抗剂,对其他 GPCR ML321 具有高选择性。在多达 168 个不同 GPCR 的功能分析筛选中,ML321 除了有效抑制 D2R 外几乎没有显示出任何活性,并且在较小程度上抑制 D3R,表现出卓越的 GPCR 选择性。 Schild 型功能测定表明 ML321 作为 D2R 的竞争性拮抗剂,而动力学研究表明 ML321 表现出缓慢开启和快速关闭的受体结合率;人们认为这些特性可以限制抗精神病药常见的锥体外系副作用。事实上,在啮齿类动物的抗精神病药物预测行为测定中使用最有效的剂量,ML321 几乎没有促进僵直症,这表明 ML321 可能对患者产生较少的锥体外系副作用。重要的是,没有其他 D2R 拮抗剂表现出这种药理学和行为特征,支持其发展成为先进的先导药物。虽然 ML321 是一种很有前途的治疗方法,但其代谢半衰期短,阻碍了其临床开发。代谢物研究表明,代谢的主要位点涉及 ML321 烷基噻吩部分的氧化。为了创造代谢更稳定的衍生物,我们迭代设计并合成了 100 多种类似物,并针对烷基噻吩部分进行了修饰。对这些类似物的 D2R 结合亲和力和功能进行了药理学表征,并测试了代谢稳定性、渗透性和溶解度。这些努力已将 ML321 优化为一系列先导候选药物,这些候选药物显示出与 ML321 相似的药理学特征,但代谢稳定性和 ADME 特性显着增加。分子对接和诱变研究使人们更好地了解 ML321 如何与 D2R 结合,这将进一步有助于模拟设计和开发。总之,这些发现增进了我们对 ML321 结构-活性关系的理解,特别是围绕烷基噻吩部分,并确定了体内药代动力学研究的主要候选者,从而代表了新抗精神病药物开发的重大进展。 自首次用于治疗帕金森病 (PD) 以来,左旋多巴一直是治疗这种疾病的黄金标准,这种疾病的定义是中枢神经系统中多巴胺能神经元进行性退化,导致严重的运动迟缓和震颤。左旋多巴的功效随着时间的推移而减弱,并与增加的副作用相关,包括运动波动和运动障碍。几种多巴胺能激动剂也被用于治疗帕金森病,包括普拉克索和罗匹尼罗,它们的运动副作用较少,但与过度赌博和性欲亢进等冲动控制障碍有关。值得注意的是,在帕金森病治疗中介导治疗作用和/或副作用的多巴胺受体亚型仍然未知。然而,普拉克索和罗匹尼罗对 D3 多巴胺受体 (D3R) 的偏好表明了这种亚型的参与,尽管这些药物在治疗剂量下也会激活 D2R。重要的是,目前用于治疗帕金森病的药物都不能改变疾病的进程,神经保护剂的发现仍然是帕金森病治疗中未满足的需求。最近,我们发现了一种新型、有效且高度选择性的 D3R 激动剂 ML417,它具有脑渗透性,并且被发现可以防止 6-OHDA 诱导的多巴胺能神经元神经变性(Moritz 等人,J. Med. Chem. 63) :5526,2020)。在当前的研究中,我们使用 ML417 来探讨 D3R 在 PD 大鼠模型中的作用。我们最初试图研究 D3R 在改善由 6-OHDA 输注至内侧前脑束诱导的偏侧帕金森病大鼠模型中运动迟缓中的作用。使用经过验证的圆柱形跑步机运动测试,根据半帕金森病大鼠的步数评估,最高 20 mg/kg 的 ML417 剂量对改善行走损伤没有影响。相比之下,给予 L-DOPA (6 mg/kg) 显着改善同一模型的运动能力。此外,用 D3R 选择性拮抗剂 SB277011A (30 mg/kg) 进行预处理并没有减弱 L-DOPA 减轻运动迟缓的作用。这些结果表明 D3R 不介导当前 PD 治疗的抗运动迟缓作用。然而,我们假设 D3R 刺激可能有益于治疗 PD 中 L-DOPA 引起的运动障碍 (LID)。为了测试这一点,我们使用慢性 L-DOPA 给药模式(12 mg/kg/天,持续 7 天)来诱导偏侧帕金森病大鼠运动障碍。随后,使用异常不随意运动 (AIM) 评分方法评估 ML417 和 SB277011A 在这些动物中的作用。单剂量 ML417 (20 mg/kg) 预处理可显着降低单剂量 L-DOPA (6 mg/kg) 引起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 的抗运动障碍作用,表明这种益处是 D3R 介导的。总体而言,这项研究表明 D3R 刺激对 PD 运动迟缓没有治疗作用,但可能有益于治疗 L-DOPA 治疗引起的运动障碍。

项目成果

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David Sibley其他文献

David Sibley的其他文献

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{{ truncateString('David Sibley', 18)}}的其他基金

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
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    8342195
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    9358531
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Dopamine Receptor Proteomics
多巴胺受体蛋白质组学
  • 批准号:
    8149634
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    10018400
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Dopamine Receptor Proteomics
多巴胺受体蛋白质组学
  • 批准号:
    7735316
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    9563096
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    7969514
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:
Molecular And Pharmacological Studies Of Dopamine Receptors
多巴胺受体的分子和药理学研究
  • 批准号:
    8149624
  • 财政年份:
  • 资助金额:
    $ 291.75万
  • 项目类别:

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