Structural basis for the functions of dopamine receptors, neurotransmitter transporters, and sigma 1 receptor

多巴胺受体、神经递质转运蛋白和 sigma 1 受体功能的结构基础

• 批准号: 10267556
• 负责人: Lei Shi
• 金额: $ 157.85万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 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/10267556
• 关键词: Adrenergic Agonists; Adrenergic Receptor; Adverse effects; Affect; Affinity; Agonist; Allosteric Site; Amphetamines; Amyotrophic Lateral Sclerosis; Antidepressive Agents; Arrestins; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biophysics; Brain; C-terminal; CNR1 gene; Cannabis; Carrier Proteins; Characteristics; Citalopram; Cocaine; Complex; Coupled; Couples; Coupling; Crystallization; Crystallography; DRD2 gene; Detergents; Development; Dissociation; Dopamine; Dopamine Receptor; Drug Targeting; Environment; Exhibits; Functional disorder; G protein-coupled inwardly-rectifying potassium channel; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Heroin; Hippocampus (Brain); Human; Hydrophobicity; Imipramine; Impairment; In Vitro; Incidence; Indoles; Investigation; Lead; Ligand Binding; Ligand Binding Domain; Ligands; Mediating; Modeling; Modification; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; N-terminal; Nervous system structure; Neurons; Neurotransmitters; Overdose; Pattern; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Play; Positioning Attribute; Protein Conformation; Proteins; Protocols documentation; Rattus; Reporting; Research; Research Subjects; Risperidone; Role; Serotonin; Serotonin Receptor 5-HT1A; Signal Transduction; Signaling Protein; Site; Structure; Structure-Activity Relationship; Substance Use Disorder; Surface; Temperature; Testing; Tetrahydrocannabinol; Therapeutic; Therapeutic Intervention; Toxic effect; Transducers; Treatment Efficacy; Vestibule; Western Blotting; addiction; base; conformational conversion; design; dopamine transporter; drug discovery; drug of abuse; eticlopride; extracellular; in silico; in vivo; inhibitor/antagonist; insight; migration; nervous system disorder; non-cannabinoid; novel; positive allosteric modulator; preservation; radioligand; receptor; recruit; response; serotonin transporter; sigma-1 receptor; simulation; small molecule; synergism; synthetic cannabinoid; synthetic opioid; therapeutic development; therapy development; trend; virtual

Aim 1. Structure-function investigations of G-protein coupled receptors By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R) revealed by crystallography, we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands. We have previously identified a G protein-biased agonist of D2R that exhibits impaired -arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. In the new study, we show that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a micro-switch for regulating receptor interactions with -arrestin. As this residue is relatively conserved among class A GPCRs, we constructed analogous mutations within other GPCRs and found that these alterations similarly impaired -arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active state structure of the 2-adrenergic receptor (2R), to build 2R-WT and 2R-Y1995.38A models in complex with the full 2R agonist BI-167107 for MD simulations. These analyses identified conformational rearrangements in 2R-Y1995.38A that propagate from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in 2R-Y1995.38A, which is predicted to affect its interactions with -arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions resulting in biased signaling. When compared to cannabis, the misuse of SCs is associated with a higher incidence of serious adverse effects, suggesting the possible involvement of non-cannabinoid sites of action. Here, we find that indole-based SCs, AM2201 and JWH-018, display minimal orthosteric interaction with several serotonin (5-HT), dopamine, and adrenergic receptors, but act as positive allosteric modulators (PAMs) at the 5-HT1A receptor (5-HT1AR), which is not observed with 9-tetrahydrocannabinol. This suggests that certain biological effects of SCs might involve allosteric interactions with 5-HT1ARs. To test this hypothesis, we examined effects of AM2201 on 5-HT1AR agonist-activated G protein-coupled inwardly-rectifying potassium channel currents and the hypothermic response to 5-HT1AR stimulation in mice lacking the cannabinoid receptor 1. We found that both 5-HT1AR-mediated responses were potentiated by AM2201, suggesting that PAM activity at 5-HT1AR may represent a novel non-cannabinoid receptor mechanism underlying their adverse effects. Aim 2. Structural basis of atypical and allosteric modulators of DAT and SERT For SERT, it has been long known that there exists a low-affinity allosteric binding site for an inhibitor S-citalopram. Binding to the allosteric site impedes dissociation of antidepressants from the high-affinity site, which may enhance antidepressant efficacy. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). Our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, a novel SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. Mutagenesis of selected Lu AF60097 interacting residues in this pose reduces its allosteric potency. Importantly, the in vivo allosteric inhibition of SERT by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we show the molecular mechanism of the allosteric coupling between S1 and S2 binding and the first lead compound towards developing high-affinity SERT allosteric inhibitors. Our findings suggest that the role of the extracellular vestibule is evolutionarily conserved among NSS as a druggable binding site for small-molecule ligands, and can be exploited for drug discovery. Aim 3. Molecular mechanisms of 1R homomerization Western blot assays have been widely used to investigate protein oligomerizations. However, the unique topology of 1R renders several intertwined challenges in western blot. We have developed a western blot protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of 1R. Using this approach, we observed an unexpected ladder-like incremental migration pattern of 1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of the intact 1R construct and the C-terminally tagged 1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged 1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impact of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies. The E102Q mutation of 1R has been found to elicit familial cases of amyotrophic lateral sclerosis. Despite reports of its downstream signaling consequences, the mechanistic details of the functional impact of E102Q at the molecular level are not clear. We investigated the molecular mechanism of the E102Q mutation with a spectrum of biochemical, biophysical, and pharmacological approaches. Our analysis of the interaction network of 1R indicates that a set of residues near E102 is critical for the integrity of the C-terminal ligand-binding domain. However, this integrity is not affected by the E102Q and E102A mutations, which is confirmed by the radioligand binding results. Instead, the E102 mutations disrupt the connection between the C-terminal domain and the N-terminal transmembrane helix (NT-helix). Results from BRET and western blot assays demonstrate that these mutations destabilize higher-order 1R oligomers, while our MD simulations based on a 1R crystal structure reveal a potential mechanism by which the mutations perturb the NT-helix dynamics. Thus, we propose that E102 is at a critical position in propagating the effects of ligand binding from the C-terminal domain to the NT-helix, while the latter may be involved in forming alternative oligomer interfaces, separate from the trimer interface in the crystal structures of 1R. Together, these results provide the first account of the molecular mechanism of 1R dysfunction caused by E102Q.

目标 1. G 蛋白偶联受体的结构功能研究 通过分析和模拟晶体学揭示的高度同源的多巴胺D2和D3受体（D2R和D3R）的非活性构象，我们发现艾氯必利以与D3R/艾氯必利结构非常相似的姿势结合D2R，但与D2R/利培酮结构不相容。此外，利培酮占据了 Na+ 结合位点附近的一个子口袋，而艾氯必利则没有。基于这些发现和我们的实验结果，我们提出Na+敏感的艾氯必利和Na+不敏感的利培酮稳定的不同受体构象对应于不同程度的反向激动。此外，我们的模拟表明，细胞外环是高度动态的，细胞外环2从D2R/利培酮结构中的螺旋构象自发转变为类似于D3R/艾氯必利结构中的扩展构象。我们的结果揭示了 D2R 非活性构象中以前未被认识到的多样性和动态。这些发现对于合理的药物发现至关重要，因为将虚拟屏幕限制为单一构象会错过相关配体。 我们之前已经鉴定出一种 G 蛋白偏向的 D2R 激动剂，其表现出β抑制蛋白募集受损。预计这种信号偏向是由配体与 D2R 的第二个细胞外环和第五个跨膜片段界面处的疏水袋之间的独特相互作用引起的。在新的研究中，我们表明该口袋内的残基 Phe189（使用 Ballesteros-Weinstein 编号的位置 5.38）充当微开关，用于调节受体与抑制蛋白的相互作用。由于该残基在 A 类 GPCR 中相对保守，因此我们在其他 GPCR 中构建了类似的突变，并发现这些改变在维持 G 蛋白信号传导的同时同样损害了抑制蛋白的募集。为了研究这种信号偏向的机制，我们使用 2-肾上腺素能受体 (2R) 的活性状态结构来构建与完整 2R 激动剂 BI-167107 复合的 2R-WT 和 2R-Y1995.38A 模型，用于 MD 模拟。这些分析鉴定了2R-Y1995.38A中的构象重排，其从细胞外配体结合位点传播到细胞内表面，导致2R-Y1995.38A中第二个细胞内环的方向改变，预计这会影响其与α抑制蛋白的相互作用。我们的研究结果为配体结合位点改变如何以变构方式影响 GPCR-转导器相互作用从而导致信号偏向提供了结构基础。 与大麻相比，滥用 SC 会导致严重不良反应的发生率更高，这表明可能涉及非大麻素的作用位点。在这里，我们发现基于吲哚的 SC AM2201 和 JWH-018 与几种血清素 (5-HT)、多巴胺和肾上腺素能受体表现出最小的正位相互作用，但在 5-HT1A 受体 (5-HT1AR) 上充当正变构调节剂 (PAM)，而使用 9-四氢大麻酚则没有观察到这种作用。这表明 SC 的某些生物学效应可能涉及与 5-HT1AR 的变构相互作用。为了检验这一假设，我们检查了 AM2201 对 5-HT1AR 激动剂激活的 G 蛋白偶联内向整流钾通道电流的影响以及缺乏大麻素受体 1 的小鼠对 5-HT1AR 刺激的低温反应。我们发现 AM2201 增强了这两种 5-HT1AR 介导的反应，这表明 5-HT1AR 的 PAM 活性可能代表了一种新的机制。 其不良影响背后的非大麻素受体机制。 目标 2. DAT 和 SERT 的非典型和变构调节剂的结构基础 对于SERT，人们早就知道抑制剂S-西酞普兰存在低亲和力变构结合位点。与变构位点的结合阻止抗抑郁药从高亲和力位点解离，这可能会增强抗抑郁药的功效。 SERT晶体结构揭示了中央结合（S1）位点和细胞外前庭（S2位点）的两个S-西酞普兰结合袋。我们的体外和计算机分析相结合表明，S1 中结合的 S-西酞普兰或丙咪嗪通过改变蛋白质构象与结合 S2 的配体变构偶联。值得注意的是，一种新型SERT抑制剂Lu AF60097是本文报道和表征的第一个高亲和力S2配体，它通过类似的机制将配体与S1结合变构偶联。在此姿势中选定的 Lu AF60097 相互作用残基的诱变降低了其变构效力。重要的是，Lu AF60097 对 SERT 的体内变构抑制通过大鼠中丙咪嗪结合的增强和海马血清素水平的增加得到证实。我们共同展示了 S1 和 S2 结合之间变构偶联的分子机制以及开发高亲和力 SERT 变构抑制剂的第一个先导化合物。我们的研究结果表明，细胞外前庭的作用在 NSS 中作为小分子配体的可成药结合位点在进化上是保守的，并且可用于药物发现。 目标 3. 1R 均聚化的分子机制 蛋白质印迹测定已广泛用于研究蛋白质寡聚化。然而，1R 独特的拓扑结构给蛋白质印迹带来了一些相互交织的挑战。我们开发了一种无需温度变性的蛋白质印迹方案来研究配体结合对 1R 寡聚状态的影响。使用这种方法，我们观察到了 1R 的意外梯状增量迁移模式，证明了洗涤剂环境中保留的同聚相互作用。我们比较了完整的 1R 构建体和 C 末端标记的 1R 构建体的迁移模式，发现对药物治疗的反应有相似的趋势。相反，N 末端标记的 1R 构建体显示出与完整构建体相反的趋势，表明寡聚化中配体结合效应的引发扭曲。总之，我们的研究结果表明，N 末端在引发结合配体的影响方面发挥着重要作用，而 C 末端适合生化研究的修饰。 已发现 1R 的 E102Q 突变会引发肌萎缩侧索硬化症家族病例。尽管有关于其下游信号转导后果的报道，但 E102Q 在分子水平上的功能影响的机制细节尚不清楚。我们通过一系列生化、生物物理和药理学方法研究了 E102Q 突变的分子机制。我们对 1R 相互作用网络的分析表明，E102 附近的一组残基对于 C 端配体结合结构域的完整性至关重要。然而，这种完整性不受 E102Q 和 E102A 突变的影响，放射性配体结合结果证实了这一点。相反，E102 突变破坏了 C 端结构域和 N 端跨膜螺旋（NT 螺旋）之间的连接。 BRET 和蛋白质印迹测定的结果表明，这些突变使高阶 1R 寡聚体不稳定，而我们基于 1R 晶体结构的 MD 模拟揭示了突变扰乱 NT 螺旋动力学的潜在机制。因此，我们认为E102在将配体结合作用从C端结构域传播到NT螺旋方面处于关键位置，而后者可能参与形成替代的寡聚物界面，与1R晶体结构中的三聚体界面分开。总之，这些结果首次解释了 E102Q 引起的 1R 功能障碍的分子机制。