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

多巴胺受体、多巴胺转运蛋白和 sigma 1 受体功能的结构基础

• 批准号: 9549754
• 负责人: Lei Shi
• 金额: $ 150.78万
• 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/9549754
• 关键词: Adverse effects; Affinity; Agonist; Animal Model; Arrestins; Back; Behavioral; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Biophysics; Carrier Proteins; Cell membrane; Cells; Characteristics; Client; Cocaine; Cocaine Abuse; Collaborations; Communication; Computational Technique; Computer Simulation; Crystallization; DRD2 gene; Data; Designer Drugs; Development; Dopamine; Dopamine D2 Receptor; Dopamine Receptor; Drug Addiction; Drug Receptors; Drug Targeting; Drug abuse; Drug usage; Endoplasmic Reticulum; Family; Functional disorder; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Goals; In Vitro; Integral Membrane Protein; Ion Channel; Lead; Learning; Ligand Binding; Ligands; Mediating; Membrane; Membrane Proteins; Memory; Mental disorders; Methamphetamine; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Models; Molecular Target; Motor; Movement; Neurons; Neurotransmitters; Operating System; Opioid; Overdose; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphotransferases; Physiological; Play; Procedures; Property; Proteins; Psychostimulant dependence; Recycling; Regulation; Research; Research Infrastructure; Resolution; Rewards; Role; Sampling; Self Administration; Serotonin; Side; Signal Pathway; Site; Sleep; Sodium; Specificity; Structure; Structure-Activity Relationship; Subgroup; Substance Use Disorder; Surface; Therapeutic; Therapeutic Intervention; Treatment Efficacy; base; biophysical techniques; cognitive reappraisal; computational chemistry; dopamine transporter; drug development; drug of abuse; ecstasy; emotion regulation; experimental study; inhibitor/antagonist; insight; interest; member; men who have sex with men; method development; models and simulation; molecular dynamics; molecular modeling; nervous system disorder; neuropsychiatry; neurotransmission; neurotransmitter release; novel; novel therapeutics; pleasure; presynaptic neurons; protein function; receptor; research and development; response; screening; serotonin transporter; sigma-1 receptor; simulation; stimulant abuse; symporter; synergism; therapeutic development; therapy development; trend

Dopamine Receptors Dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions abnormalities in its regulation have been implicated in neuropsychiatric and substance use disorders. Dopamine receptors belong to G protein-coupled receptor (GPCR) family. The members of dopamine D2-like receptor subgroup (consisting of D2R, D3R, and D4R) are implicated in various vital physiological functions, including voluntary movement, reward, sleep, memory, learning, and pleasure. That D3R expression is elevated in response to drugs of abuse, has prompted efforts toward the development of D3R-selective agents for the treatment of drug addiction. Inhibition of D3R may be less prone to causing motor side effects that can result from D2R blockade. Currently we focus on investigating the intermediate conformational states between the crystallographically identified inactive and active states, and the changes in the interaction network that mediate the transitions among these states, in order to reveal the structural basis for the partial agonism of selected ligands, the allosteric property of a negative allosteric modulator (NAM) and the of Na+ sensitivity of selected antagonists and NAM. By using the experimental functional assays being adapted and developed in my group (see Method Development), we will achieve efficient synergy between the in vitro and in silico characterizations of i) ligand binding in D3R, D2R, and other dopamine receptors, and ii) the functional crosstalks between receptors and different signaling pathways, i.e., G-protein vs. -arrestin, and the selectivity among various types of G-proteins. Such efforts, in close collaboration with medicinal chemists, will lead towards achieving the goal of tailoring desired specificity and efficacy, not only for the receptor subtypes, but also for the targeted signaling pathways. Dopamine and Serotonin Transporters DAT and SERT belong to the Neurotransmitter:Sodium Symporter (NSS) family, and serve to terminate dopamine and serotonin neurotransmission respectively, by recycling released neurotransmitters back into the presynaptic neuron. DAT is the primary target for abused psychostimulants such as cocaine and methamphetamine, whereas 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) binds to SERT. An understanding of the full spectrum of functional states and their transitions in a transporter cycle is required to understand the functions of these proteins and the complexity of the ligand binding modes, in order to identify and eventually exploit the therapeutic opportunities in reducing the efficacy of the abused drugs. Thus, the varied inhibition mechanisms of inhibitors are of particular interest in developing targeted and effective therapeutic interventions for drug abuse and other psychiatric disorders. Currently we focus on investigating the structural basis of the atypical DAT inhibitors that may stabilize the transporter in inward-facing conformations, as well as the molecular mechanisms of the allosteric modulations of NSS by ligands targeting the EV, using MhsT and SERT as model systems. Similar to targeting the SBPs of D3R and D2R (see above), allosteric modulation achieved by targeting the S2 site has obvious advantages over competitive inhibitions of substrate binding in the canonical S1 site, in terms retaining the normal functions of DAT and SERT. The resulting molecular models stably bound with either S2:substrate or S2:inhibitor will serve to optimize the allosteric inhibitors. Indeed, in collaboration with medicinal chemists, we have begun to identify novel SERT S2-specific inhibitors with even higher affinities. Our mechanistic understanding of the allosteric communications between the S1 and S2 sites will guide to further refine the allosteric inhibition properties, e.g., either sensitive or insensitive to the ligand bound in the S1 site. Sigma 1 Receptor The sigma 1 receptor (1R) is a structurally unique transmembrane protein that functions as a molecular chaperone. It is located at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), and has been found to translocate to the plasma membrane and other parts of the cell, and modulates the functions of a number of ion channels, receptors, or kinases, including targets relevant to drug abuse such as dopamine receptors and DAT. Dysfunctions of such modulations are connected to many neurological disorders. In particular, 1R has been implicated in cocaine abuse. Cocaine shows biochemical affinity and pharmacological activity at both DAT and 1R. It has been demonstrated that compounds that can antagonize the action of cocaine at both sites may have therapeutic potential of cocaine abuse. The behavioral effects of the 1R drugs in the animal models of psychostimulant dependence are quite remarkable 1R agonists can substitute for cocaine in self-administration, and the antagonists can block the self-administration interestingly, a few DAT inhibitors have high affinities for both DAT and 1R (e.g., those described in and aforementioned JHW007). However, the 1R pharmacology and the synergy of the ligands on other targets, in particular DAT, are ill-defined at molecular and cellular levels. Currently we focus on using a combined experimental and computational approach to study the molecular interactions between 1R and its ligands based on the newly available crystal structure. We use molecular modeling and simulations and experimental molecular biophysics approaches to characterize ligand-induced conformational and oligomerizational changes of 1R, and to investigate its interactions with the client proteins that have high-resolution structural information, such as BiP and DAT. Method Development The research on drug abuse needs to accommodate new drugs and drug use trends, such as the designer drugs that contribute to the current opioid crisis. In order to develop therapeutic and/or overdose reversal procedures, efficient characterizing the efficacy of abused compounds requires adequate mechanistic understanding of the usual targets of the abused drugs, and the functional crosstalk among these targets from molecular and cellular levels. Thus, in addition to contributing to the corresponding scientific fields in general, the computational and experimental approaches being developed below will serve to streamline the characterization of the drug-protein interactions, to identify related mechanism features, and to eventually establish mechanistic interaction framework of the targets. MD simulations is the basic computational technique we are using to rigorously characterize ligand-protein interactions. While we have been using the MSM analysis to integrate the simulations data together, we have just started to guide MD simulations with MSM to efficiently sample the energy surfaces by avoiding well-sampled regions. Although the MSM-based adaptive sampling has been proposed, no infrastructure is publicly available, and such an infrastructure depends on the operating system setup and hardware composition. Thus, we are establishing an automated procedure to identify the under-sampled microstates for any given simulated condition from MSM analysis as the starting point for additional MD simulations. This is part of our efforts towards the long-term goal of high-throughput MD to efficiently study (ligand-induced) multiple conformational states of a protein target.

多巴胺受体 多巴胺在认知、情绪和行为功能的调节中发挥着重要作用，其调节异常与神经精神和物质使用障碍有关。多巴胺受体属于 G 蛋白偶联受体 (GPCR) 家族。多巴胺 D2 样受体亚组（由 D2R、D3R 和 D4R 组成）的成员与多种重要的生理功能有关，包括随意运动、奖励、睡眠、记忆、学习和愉悦。 D3R 表达因滥用药物而升高，促使人们努力开发用于治疗药物成瘾的 D3R 选择性药物。抑制 D3R 可能不太容易引起因 D2R 阻断而导致的运动副作用。 目前，我们重点研究晶体学鉴定的非活性和活性状态之间的中间构象状态，以及介导这些状态之间转变的相互作用网络的变化，以揭示所选配体部分激动的结构基础、负变构调节剂（NAM）的变构特性以及所选拮抗剂和NAM的Na+敏感性。 通过使用我的小组正在调整和开发的实验功能测定（参见方法开发），我们将在 i）D3R、D2R 和其他多巴胺受体中的配体结合，以及 ii）受体和不同信号通路（即 G 蛋白与 -arrestin）之间的功能串扰以及各种类型的选择性之间的体外和计算机表征之间实现有效的协同作用。 G-蛋白。这些努力与药物化学家密切合作，将实现定制所需特异性和功效的目标，不仅针对受体亚型，而且针对目标信号传导途径。 多巴胺和血清素转运蛋白 DAT 和 SERT 属于神经递质：钠转运蛋白 (NSS) 家族，通过将释放的神经递质回收回突触前神经元，分别终止多巴胺和血清素神经传递。 DAT 是可卡因和甲基苯丙胺等滥用精神兴奋剂的主要目标，而 3,4-亚甲二氧基甲基苯丙胺（MDMA、摇头丸）则与 SERT 结合。需要了解转运蛋白循环中的全部功能状态及其转变，以了解这些蛋白质的功能和配体结合模式的复杂性，以便识别并最终利用降低滥用药物功效的治疗机会。因此，抑制剂的不同抑制机制对于开发针对药物滥用和其他精神疾病的有针对性的有效治疗干预措施特别有意义。 目前，我们重点研究非典型 DAT 抑制剂的结构基础，这些抑制剂可以稳定转运蛋白的内向构象，以及使用 MhsT 和 SERT 作为模型系统，通过针对 EV 的配体对 NSS 进行变构调节的分子机制。 与靶向 D3R 和 D2R 的 SBP（见上文）类似，通过靶向 S2 位点实现的变构调节在保留 DAT 和 SERT 的正常功能方面比经典 S1 位点中底物结合的竞争性抑制具有明显的优势。由此产生的与 S2: 底物或 S2: 抑制剂稳定结合的分子模型将有助于优化变构抑制剂。事实上，通过与药物化学家合作，我们已经开始鉴定具有更高亲和力的新型 SERT S2 特异性抑制剂。我们对 S1 和 S2 位点之间变构通讯的机制理解将指导进一步完善变构抑制特性，例如对 S1 位点中结合的配体敏感或不敏感。 西格玛1受体 sigma 1 受体 (1R) 是一种结构独特的跨膜蛋白，充当分子伴侣。它位于线粒体相关内质网 (ER) 膜 (MAM)，并被发现可易位至质膜和细胞的其他部分，并调节许多离子通道、受体或激酶的功能，包括与药物滥用相关的靶标，如多巴胺受体和 DAT。这种调节功能障碍与许多神经系统疾病有关。特别是，1R 与可卡因滥用有关。可卡因在 DAT 和 1R 上均显示出生化亲和力和药理活性。已经证明，能够在两个位点拮抗可卡因作用的化合物可能具有治疗可卡因滥用的潜力。 1R药物在精神兴奋剂依赖的动物模型中的行为效应是相当显着的，1R激动剂可以替代可卡因自我给药，有趣的是拮抗剂可以阻断自我给药，一些DAT抑制剂对DAT和1R都具有高亲和力（例如前面提到的JHW007中描述的那些）。然而，1R 药理学和配体对其他靶点（特别是 DAT）的协同作用在分子和细胞水平上尚不明确。 目前，我们专注于使用实验和计算相结合的方法，基于新获得的晶体结构来研究 1R 及其配体之间的分子相互作用。我们使用分子建模和模拟以及实验分子生物物理学方法来表征配体诱导的 1R 构象和寡聚变化，并研究其与具有高分辨率结构信息的客户蛋白（例如 BiP 和 DAT）的相互作用。 方法开发 关于药物滥用的研究需要适应新药物和药物使用趋势，例如导致当前阿片类药物危机的设计药物。为了开发治疗和/或过量逆转程序，有效表征滥用化合物的功效需要对滥用药物的常见靶点以及这些靶点之间从分子和细胞水平的功能串扰有充分的机制理解。因此，除了总体上对相应的科学领域做出贡献之外，下面开发的计算和实验方法将有助于简化药物-蛋白质相互作用的表征，识别相关的机制特征，并最终建立靶标的机械相互作用框架。 MD 模拟是我们用来严格表征配体-蛋白质相互作用的基本计算技术。虽然我们一直在使用 MSM 分析将模拟数据集成在一起，但我们才刚刚开始使用 MSM 指导 MD 模拟，通过避免采样良好的区域来有效地对能量表面进行采样。尽管已经提出了基于MSM的自适应采样，但没有公开可用的基础设施，并且这种基础设施取决于操作系统设置和硬件组成。因此，我们正在建立一个自动化程序，以从 MSM 分析中识别任何给定模拟条件下的欠采样微观状态，作为其他 MD 模拟的起点。这是我们为实现高通量 MD 的长期目标而努力的一部分，以有效地研究（配体诱导的）蛋白质靶标的多种构象状态。