生物谷综合：目前，全球有众多抑郁症者患者依赖抗抑郁药缓解痛苦。但是关于抗抑郁药的作用机制却一直没有弄清楚。近日，美国两个独立研究小组分别攻克了这一难题，这对抑郁症患者来说无疑是个鼓舞人心的好消息。相关论文分别在线发表于8月8日的《自然》和8月9日的《科学》杂志上。
抗抑郁药主要作用是阻止一些特定化学物质输入脑部神经元，这些化学物质包括多巴胺、复合胺等，它们用来在细胞间传递信息。而这些化学物质的输入则依赖细胞外膜上称作转运蛋白（transporter proteins）的分子通道。抗抑郁药正是在这些分子通道上发挥它的作用。但是抗抑郁药到底是怎样发挥作用的深层机制却一直没有弄清。
为了解决这一长达45年的难题，两个独立研究小组开始了攻关行动。领导者分别是美国俄勒冈健康与科学大学（Oregon Health and Science University）的Eric Gouaux和纽约大学的Da-Neng Wang。由于人类的细胞的转运蛋白难以分离且易于分解，所以他们实验中采用的是细菌的相似蛋白——LeuT。他们通过实验令细菌转运蛋白与所选抗抑郁药（Gouaux小组所用为clomipramine，Wang小组所用为具有相似组分的desipramine）形成结晶，这样转运蛋白与抗抑郁药就“绑”在了一起。然后，研究人员应用X射线结晶学（X-ray crystallography）放大转运蛋白的结构并进行分析。
两个小组得到了相似的结果：抗抑郁药绑定在转运蛋白的外表，改变了该蛋白的结构，这就使得转运蛋白内部的诸如多巴胺等种化学物质无法进入到神经细胞中。
Gouaux表示，他的小组主要关注的是，抗抑郁药怎样保持转运蛋白内的化学物质不外泄。而现在这已基本弄清，所以下一步的工作就是设计分子来达到这一目的。
而Wang和他的小组关注的则是，怎样将这个结果推广至人类身上。他和研究小组首先找到人类转运蛋白中类似于LeuT与抗抑郁药相绑定的部位，运用基因技术使这一部位发生突变，然后加入抗抑郁药desipramine。结果发现，抗抑郁药并不能阻止细胞吸收多巴胺等化学物质，这表明抗抑郁药并不能有效阻止绑定位置经过修正的人类转运蛋白。Wang认为，这证明人类的相关抑制机制和绑定位置被隐藏了。
针对此次研究，也有不同观点。英国牛津大学的药理学家Les Iversen就对此次研究的实用性表示了怀疑。他认为，以前分子结构也没有弄清，但是有效的药物照样开发出来了。他说：“我认为我们没必要弄清这些，药物开发人员一直在研究这些绑定位置，虽然并不知道它们的具体位置。”（科学网 梅进/编译）
原始出处:
Nature advance online publication 8 August 2007 | doi:10.1038/nature06038; Received 30 January 2007; Accepted 21 June 2007; Published online 8 August 2007
Antidepressant binding site in a bacterial homologue of neurotransmitter transporters
Satinder K. Singh1, Atsuko Yamashita3,4 & Eric Gouaux1,2
The Vollum Institute and,
Howard Hughes Medical Institute, Oregon Health and Science University, 3181 S.W. Sam Jackson Road, Portland, Oregon 97239, USA
Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA
Present address: RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan.
Correspondence to: Eric Gouaux1,2 Correspondence and requests for materials should be addressed to E.G. (Email: gouauxe@ohsu.edu).
Abstract
Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer1. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption2, congenital hypothyroidism3, Bartter's syndrome4, epilepsy5, depression6, autism7 and obsessive-compulsive disorder8. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics9, anticonvulsants10 and antidepressants11, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive12, 13 and noncompetitive14, 15 modes of inhibition exist. Antagonist dissociation experiments on a serotonin transporter tricyclic antidepressant have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site16. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine binds in an extracellular-facing vestibule about 11 Å above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.
