Structural Biology of Dopamine Signaling

摘要 人多巴胺信号通路诱导和促进多巴胺神经传递 中脑边缘多巴胺能通路，它改变了与奖励相关的行为，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具有建设性 受多巴胺受体(DRS)调节。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目标。 尽管人类DR(HDR)家族成员中的一些受体激动剂(例如，多巴胺D1样) 受体)正在被深入研究以进行治疗干预，但其成功受到了极大的阻碍 由于依从性和有效性较差，或由于相关副作用。与此相关的是，药物学的多元药理学 最近的研究发现了多巴胺D1样受体和其他HDR。基于新知识的 D1R和D5R样受体的结构不仅揭示了它们的信号机制，而且还提供了 可用于促进合理药物设计的新理解。 HDRs属于G蛋白偶联受体(GPCR)家族，以生成困难而臭名昭著 X射线测定高分辨结构所必需的衍射级晶体 结晶学。这项研发建议旨在为结构和技术开发一个全面和强大的平台 多巴胺D_1样受体及其与G蛋白的复合体的功能研究 以这些受体为靶标的亲和纳米体、抗体和配体。这个平台将涉及多个步骤 它们通过前向和后向反馈系统紧密地相互连接和循环。私家侦探现在 还与其他具有不同专业知识的研究小组建立了强大的合作关系 方法包括X射线自由电子激光、微晶电子衍射技术等。 提出了三个具体目标：(1)人D1R(HD1R)在非活动状态下的结构/功能研究，(2) 利用X射线结晶学和计算生物学方法研究hD1R的可能活性状态， (3)建立确定hD1R与G蛋白络合物结构的优化方法 使用EM映像的合作伙伴。本研究对多巴胺信号转导途径的意义是多方面的。 相关药物发现研究：1)我们将深入了解多巴胺D1样受体的功能和 变构调节，2)我们将能够广泛地筛选，以确定新的高亲和力的HDRs配体，3) 不同多巴胺受体间DR信号转导和配体选择机制的研究 亚家族，4)刺激基于HDR结构的药物设计，5)研究HDRs/G蛋白复合体信号转导和 揭示了激活机理，为低温电磁技术在疑难问题上的应用铺平了道路 膜蛋白是未来的靶标。

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.