去甲肾上腺素（Noradrenaline, NA/NE）作为一种关键的神经递质，对于调整大脑和身体进入活跃状态具有至关重要的作用。去甲肾上腺素系统的功能紊乱可导致多种疾病，包括交感神经过度激活、注意力缺陷与多动障碍（Attention Deficit and Hyperactive Disorder , ADHD）、自主神经功能衰竭、应激、抑郁和嗜铬细胞瘤等。去甲肾上腺素转运体（Noradrenaline transporter, NET）是存在于去甲肾上腺素能神经元突触前膜的一类跨膜转运体，属于溶质载体转运蛋白家族6（Solute carrier transporter family 6, SLC6）的一员，主要负责将NA主动转运回突触前末梢，进而调控NA信号的作用强度和时间。

突触间隙中80-90%的NA由NET重新摄取回神经末梢，是去甲肾上腺素能系统的主要调控蛋白。因此，以NET为靶点的抑制剂是治疗抑郁症和ADHD等精神类疾病的有效策略。目前临床上用于治疗抑郁症的药物主要作用于5-羟色胺转运蛋白（Serotonin transporter, SERT）, 而特异性抑制NET的药物相对较少，造成这一现象的主要原因是NET三维结构以及与特异性抑制剂相互作用位点信息的缺失，导致研究人员无法合理设计以NET为靶点的特异性抑制剂。雷达法辛（Radafaxine, RDF）是非典型性抗抑郁药——安非他酮的一种强效代谢物，能够有效地抑制NET的底物转运功能。相比于安非他酮，雷达法辛对NET具有更高的亲和力，是研究NET特异性抑制剂结合位点的理想工具。基于此，本研究利用单颗粒冷冻电镜技术成功解析了人源NET（Human noradrenaline transporter, hNET）在无底物结合状态和雷达法辛结合状态下的三维结构，分辨率分别为2.9 Å和3.0 Å。通过对结构的进一步解析以及与其他构象状态下的同源蛋白结构的比对，本研究揭示了雷达法辛与NET的相互作用位点，阐明其通过氢键、离子键和疏水作用等稳定地结合在底物结合口袋，并且通过阻碍NET由向内开放到向外开放（Outward-facing conformation）的构象转变而发挥抑制作用。

NET的异常高表达与多种癌症具有密切的相关性，包括神经母细胞瘤、嗜铬细胞瘤和结直肠癌等，以NET为靶标的底物类似物在癌症靶向诊断和治疗领域具有重要的意义。间碘苄胍（Meta-iodobenzylguanidine, MIBG）是去甲肾上腺素的合成胍乙啶类似物，能够被NET特异性识别并转运，其放射性标记物——¹²³I-MIBG已被广泛用于多种神经内分泌肿瘤的成像诊断，被认为是神经母细胞瘤患者诊断、分期和随访的金标准，具有重要的临床意义。此外，基于¹²³I-MIBG良好的肿瘤靶向和杀伤作用，已被FDA批准用于部分肿瘤治疗的保守方案。因此，NET与MIBG关键结合位点以及转运模式的阐明不仅能够反映生理状态下NET对于特异性底物NA的摄取机制，弥补这一领域基础知识的空缺，更为后续肿瘤诊断以及治疗药物的研发提供理论基础。基于此，本研究利用冷冻电镜成功解析人源NET蛋白与MIBG结合状态下的三维结构，分辨率为2.8 Å，并进一步揭示了MIBG与NET的关键结合位点。

综上所述，在本课题开展之前，尽管NET在生物学和临床上的重要性已得到确认，但由于未能获得NET蛋白在底物结合和抑制剂结合状态下的高分辨率结构，导致其底物运输和药物抑制的具体分子机制仍不清楚，这大大限制了新型NET底物类似物和抑制剂的开发。利用单颗粒冷冻电镜技术，本研究不仅揭示了人源NET的整体架构及其底物结合模式，也阐明了非典型抗抑郁药安非他酮的活性代谢物——雷达法辛的抑制机理，为进一步理解hNET的工作机制以及后期基于结构的新型化合物的开发提供了重要的理论基础。

Noradrenaline (NA/NE) is a crucial neurotransmitter that plays a central role in modulating brain and body activity. Dysfunction of the noradrenergic system has been implicated in a range of disorders, including excessive sympathetic activation, attention deficit hyperactivity disorder (ADHD), autonomic dysfunction, stress, depression, and pheochromocytoma.

The noradrenaline transporter (NET), a member of the solute carrier family 6 (SLC6), is a transmembrane protein located on the presynaptic membrane of noradrenergic neurons. It is primarily responsible for the reuptake of approximately 80–90% of NA from the synaptic cleft back into presynaptic terminals, thereby regulating the intensity and duration of noradrenergic signaling. As an important regulator of NA homeostasis, NET represents a promising target for the treatment of psychiatric and neurological disorders. Inhibitors of NET have demonstrated therapeutic efficacy in treating conditions such as depression and ADHD. However, most clinically used antidepressants predominantly target the serotonin transporter (SERT), with relatively few drugs specifically targeting NET. A major limiting factor in the development of selective NET inhibitors has been the lack of high-resolution structural information regarding NET and its interaction with specific ligands. Radafaxine (RDF), a potent metabolite of the atypical antidepressant bupropion, exhibits higher affinity for NET compared to its parent compound and serves as an ideal tool for investigating the binding mechanisms of NET-specific inhibitors. In this study, the three-dimensional structures of human NET in both its native state and radafaxine-bound state were successfully determined using cryo-electron microscopy (cryo-EM), achieving resolutions of 2.9 Å and 3.0 Å, respectively. Structural analysis, combined with comparisons to homologous transporters in different conformational states, revealed that radafaxine binds stably within the substrate-binding pocket through hydrogen bonds, ionic interactions, and hydrophobic contacts. This binding inhibits the conformational transition of NET from inward-facing to outward-facing state, effectively blocking NA transport.

Moreover, abnormal overexpression of NET is closely associated with several cancers, including neuroblastoma, pheochromocytoma, and colorectal cancer. Substrate analogs that target NET hold great potential in the field of cancer diagnosis and therapy. Meta-iodobenzylguanidine (MIBG), a synthetic guanethidine analog of NA, is specifically recognized and transported by NET. The radiolabeled form, ¹²³I-MIBG, has become the gold standard for imaging and monitoring neuroblastoma, playing a critical role in diagnosis, staging, and follow-up assessments. Furthermore, due to its tumor-targeting properties, MIBG has also been incorporated into certain conservative cancer treatment regimens. To better understand the molecular basis of MIBG recognition and transport by NET, this study further determined the structure of human NET in complex with MIBG at a resolution of 2.8 Å using cryo-electron microscopy. This enabled the identification of key binding sites and provided insights into the transport mechanism of MIBG, which may also reflect the physiological uptake of NA under normal conditions.

Prior to this study, despite the well-established biological and clinical importance of NET, the molecular mechanisms underlying its substrate transport and drug inhibition remained largely unknown due to the absence of high-resolution structural data. By determining the high-resolution structures of human NET in multiple functional states—native, radafaxine-bound, and MIBG-bound—this work not only reveals the structural architecture and substrate-binding modes of hNET but also elucidates the inhibitory mechanism of radafaxine. These findings provide a solid foundation for understanding the functional dynamics of NET and pave the way for the rational design of novel therapeutics targeting the noradrenergic system, as well as for developing advanced diagnostic and therapeutic agents for NET-expressing tumors.