Structural Biology of Dopamine Signaling

摘要 人多巴胺信号通路通过多巴胺受体诱导和促进多巴胺神经传递。 中脑边缘多巴胺能通路，它改变奖励相关的行为，并与 许多疾病的发展，包括精神分裂症、亨廷顿病、认知障碍和 帕金森氏症。越来越多的证据表明，这一重要的信号通路是建设性的， 多巴胺受体（Dopamine Receptor，DRs）因此，使该膜蛋白家族的成员高度 临床前和临床研究都支持有前景的治疗靶点。 尽管人DR（hDR）家族成员中的受体的一些激动剂（例如多巴胺D1样受体）是已知的，但它们在人DR（hDR）家族中的作用并不明显。 受体）正在被深入研究用于治疗干预，它们的成功受到了极大的阻碍 由于不良的依从性和功效或由于相关的副作用。与此相关， 多巴胺D1样受体和其他hDR在最近的研究中已经被发现。新知识基于 D1样受体（D1 R和D5 R）的结构不仅可以揭示它们的信号转导机制，而且可以提供 新的理解，可以利用，以促进合理的药物设计。 hDR属于G蛋白偶联受体（GPCR）家族，该家族因难以产生 衍射质量的晶体，对于通过X射线确定高分辨率结构至关重要 结晶学该研发提案旨在开发一个全面且强大的平台，用于结构和 多巴胺D1样受体和hDR与G蛋白复合物的功能研究，用于筛选高水平的 亲和纳米抗体、抗体和靶向这些受体的配体。这个平台将涉及多个步骤 它们紧密相连，并通过一个前向和后向反馈系统循环。PI现在 还与其他具有不同专业知识的研究小组建立了强有力的合作关系， X射线自由电子激光、微晶电子衍射技术等。 本文提出了三个具体目标：（1）非活性状态下人D1 R（hD 1 R）的结构/功能研究; 使用X射线晶体学和计算生物学方法来研究hD 1 R的假定活性状态， （3）建立了确定hD 1 R与G蛋白复合物结构的优化方法 使用EM成像的合作伙伴。本研究的意义是多方面的多巴胺信号通路， 相关的药物发现研究：1）我们将深入了解多巴胺D1样受体的功能， 变构调节，2）我们将能够广泛筛选，以确定新的hDR高亲和力配体，3） 描述不同多巴胺受体之间DR信号传导和配体选择的机制 亚家族，4）刺激基于hDR结构的药物设计，5）检查hDR/G蛋白复合物信号传导， 为冷冻电镜技术应用于疑难病的研究奠定了基础 膜蛋白靶点。

Abstract Human dopamine signaling pathway induces and facilitates dopamine neurotransmission through the mesolimbic dopaminergic pathway, which modifies reward-related behaviors and is associated with the development of many diseases, including schizophrenia, Huntington's disease, cognitive disorders and Parkinson’s disease. Mounting evidence suggests that this important signaling pathway is constructively regulated by the dopamine receptors (DRs). Thus, making members of this membrane protein family highly promising therapeutic targets as supported by both pre-clinical and clinical studies. Although some agonists of receptors in the human DR (hDR) family members (e.g. for dopamine D1-like receptors) are being intensively studied for therapeutic intervention, their success has been greatly hampered due to poor adherence and efficacy, or due to associated side effects. Relatedly, the polypharmacology of dopamine D1-like receptor and other hDRs have been discovered in recent studies. New knowledge based on structures of D1-like receptors (D1R and D5R) will not only reveal their signaling mechanisms, but also provide new understanding that can be exploited to facilitate rational drug design. hDRs belong to the G protein-coupled receptor (GPCR) family which is notorious for difficulties in generating diffraction-quality crystals that are essential for the determination of high-resolution structures by X-ray crystallography. This R&D proposal aims to develop a comprehensive and robust platform for structural and functional studies of dopamine D1-like receptors and complexes of hDRs with G proteins, for screening high- affinity nanobodies, antibodies, and ligands targeting these receptors. This platform will involve multiple steps that are closely interconnected and looped through a forward and backward feedback system. The PI has now also established strong collaborations with other research groups with different expertise as alternative approaches, including X-ray free electron laser, Microcrystal Electron Diffraction technique, etc. Three specific aims are proposed: (1) Structure/function studies of human D1R (hD1R) in the inactive state, (2) Using X-ray crystallography and computational biology approaches to study the putative active state of hD1R, and (3) Establishing optimized approaches for determining the structure of complexes of hD1R with G protein partners using EM imaging. The significance of this study is multi-fold on dopamine signaling pathway and related drug discovery studies: 1) we will gain insights into dopamine D1-like receptor functionalities and allosteric modulations, 2) we will be able to screen extensively to identify new high-affinity ligands for hDRs, 3) characterize the mechanisms of DR signaling and ligand selection between different dopamine receptor subfamilies, 4) stimulate hDR structure-based drug design, 5) examine hDRs/G protein complex signaling and reveal the activation mechanism, and 6) pave the road for the application of cryoEM technology on difficult membrane protein targets in the future.