生物谷綜合：目前，全球有眾多抑郁癥者患者依賴抗抑郁藥緩解痛苦,。但是關(guān)于抗抑郁藥的作用機(jī)制卻一直沒有弄清楚,。近日，美國(guó)兩個(gè)獨(dú)立研究小組分別攻克了這一難題,，這對(duì)抑郁癥患者來說無疑是個(gè)鼓舞人心的好消息,。相關(guān)論文分別在線發(fā)表于8月8日的《自然》和8月9日的《科學(xué)》雜志上,。
抗抑郁藥主要作用是阻止一些特定化學(xué)物質(zhì)輸入腦部神經(jīng)元,，這些化學(xué)物質(zhì)包括多巴胺,、復(fù)合胺等,，它們用來在細(xì)胞間傳遞信息,。而這些化學(xué)物質(zhì)的輸入則依賴細(xì)胞外膜上稱作轉(zhuǎn)運(yùn)蛋白（transporter proteins）的分子通道?？挂钟羲幷窃谶@些分子通道上發(fā)揮它的作用,。但是抗抑郁藥到底是怎樣發(fā)揮作用的深層機(jī)制卻一直沒有弄清,。
為了解決這一長(zhǎng)達(dá)45年的難題,，兩個(gè)獨(dú)立研究小組開始了攻關(guān)行動(dòng),。領(lǐng)導(dǎo)者分別是美國(guó)俄勒岡健康與科學(xué)大學(xué)（Oregon Health and Science University）的Eric Gouaux和紐約大學(xué)的Da-Neng Wang,。由于人類的細(xì)胞的轉(zhuǎn)運(yùn)蛋白難以分離且易于分解,，所以他們實(shí)驗(yàn)中采用的是細(xì)菌的相似蛋白——LeuT。他們通過實(shí)驗(yàn)令細(xì)菌轉(zhuǎn)運(yùn)蛋白與所選抗抑郁藥（Gouaux小組所用為clomipramine,，Wang小組所用為具有相似組分的desipramine）形成結(jié)晶，這樣轉(zhuǎn)運(yùn)蛋白與抗抑郁藥就“綁”在了一起,。然后,，研究人員應(yīng)用X射線結(jié)晶學(xué)（X-ray crystallography）放大轉(zhuǎn)運(yùn)蛋白的結(jié)構(gòu)并進(jìn)行分析。
兩個(gè)小組得到了相似的結(jié)果：抗抑郁藥綁定在轉(zhuǎn)運(yùn)蛋白的外表,，改變了該蛋白的結(jié)構(gòu),，這就使得轉(zhuǎn)運(yùn)蛋白內(nèi)部的諸如多巴胺等種化學(xué)物質(zhì)無法進(jìn)入到神經(jīng)細(xì)胞中。
Gouaux表示,，他的小組主要關(guān)注的是,，抗抑郁藥怎樣保持轉(zhuǎn)運(yùn)蛋白內(nèi)的化學(xué)物質(zhì)不外泄。而現(xiàn)在這已基本弄清,，所以下一步的工作就是設(shè)計(jì)分子來達(dá)到這一目的。
而Wang和他的小組關(guān)注的則是,，怎樣將這個(gè)結(jié)果推廣至人類身上,。他和研究小組首先找到人類轉(zhuǎn)運(yùn)蛋白中類似于LeuT與抗抑郁藥相綁定的部位,，運(yùn)用基因技術(shù)使這一部位發(fā)生突變,，然后加入抗抑郁藥desipramine,。結(jié)果發(fā)現(xiàn)，抗抑郁藥并不能阻止細(xì)胞吸收多巴胺等化學(xué)物質(zhì),，這表明抗抑郁藥并不能有效阻止綁定位置經(jīng)過修正的人類轉(zhuǎn)運(yùn)蛋白。Wang認(rèn)為,，這證明人類的相關(guān)抑制機(jī)制和綁定位置被隱藏了,。
針對(duì)此次研究,，也有不同觀點(diǎn),。英國(guó)牛津大學(xué)的藥理學(xué)家Les Iversen就對(duì)此次研究的實(shí)用性表示了懷疑。他認(rèn)為,，以前分子結(jié)構(gòu)也沒有弄清,，但是有效的藥物照樣開發(fā)出來了。他說：“我認(rèn)為我們沒必要弄清這些,，藥物開發(fā)人員一直在研究這些綁定位置,，雖然并不知道它們的具體位置,。”（科學(xué)網(wǎng) 梅進(jìn)/編譯）
原始出處:
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: [email protected]).
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.
