GPCR和激酶等靶標(biāo)存在較為明確的內(nèi)源性配體結(jié)合口袋，其激動(dòng)劑類藥物一般是作用于該口袋,，在一定程度上取代（模仿）內(nèi)源性激動(dòng)劑的功能,。針對(duì)GPCR和激酶開展的基于結(jié)構(gòu)的藥物設(shè)計(jì)已有很多成功案例。與這些受體和激酶不同,，電壓門控通道是被電壓激活，沒有明確的常規(guī)內(nèi)源性配體結(jié)合口袋,。確證激動(dòng)劑的作用位點(diǎn)是電壓門控通道研究領(lǐng)域的難點(diǎn)之一,，通過基于結(jié)構(gòu)的藥物設(shè)計(jì)發(fā)現(xiàn)電壓門控通道激動(dòng)劑也進(jìn)而面臨很大挑戰(zhàn)。
KCNQ2是癲癇相關(guān)的一類電壓門控鉀離子通道,。上海藥物所蔣華良課題組和神經(jīng)藥理學(xué)國(guó)際科學(xué)家工作站研究人員通過綜合運(yùn)用動(dòng)力學(xué)模擬,、分子對(duì)接、定點(diǎn)突變和電生理測(cè)試等方法,，發(fā)現(xiàn)了一個(gè)位于通道門控電荷通路(gating charge pathway)中的激動(dòng)劑結(jié)合口袋,。針對(duì)該口袋，開展了虛擬篩選和藥物設(shè)計(jì),，從20萬個(gè)化合物中挑選出25個(gè)候選分子,。經(jīng)電生理測(cè)試確認(rèn)9個(gè)KCNQ2新激動(dòng)劑，其中兩個(gè)在兩類動(dòng)物模型中表現(xiàn)出優(yōu)異的抗癲癇活性,。該研究為發(fā)現(xiàn)離子通道調(diào)制劑結(jié)合口袋提供了成功的案例,，并且首次實(shí)現(xiàn)了“基于結(jié)構(gòu)的電壓門控鉀離子通道激動(dòng)劑發(fā)現(xiàn)”,，為離子通道藥物研究領(lǐng)域的一個(gè)重要進(jìn)展,。
該研究還推動(dòng)了電壓門控通道結(jié)構(gòu)功能關(guān)系研究,，更新了人們對(duì)電壓敏感區(qū)結(jié)構(gòu)和門控機(jī)制的認(rèn)識(shí),。基于Kv1.2,、Kv1.2-2.1和Shaker等電壓門控鉀離子通道的研究,，傳統(tǒng)觀點(diǎn)認(rèn)為門控電荷通路相對(duì)狹窄，在中間部位由一個(gè)保守的苯丙氨酸及周圍殘基形成所謂“閉塞位點(diǎn)”(occluded site),，將門控電荷通路分隔成內(nèi)外兩個(gè)部分,，是“集中電場(chǎng)”和“門控電荷傳遞中心”等電壓門控離子通道電壓感受理論的結(jié)構(gòu)基礎(chǔ)。該項(xiàng)研究顯示，KCNQ2通道的相應(yīng)位置則存在一個(gè)空腔,，可容納結(jié)構(gòu)多樣性的小分子配體,，且通過突變實(shí)驗(yàn)確認(rèn)保守性苯丙氨酸在不同通道中功能存在明顯差異，因而支持門控電荷通路的結(jié)構(gòu)存在多樣性,。
6月25日,，Cell Research在線發(fā)表了該研究結(jié)果。論文共同第一作者為上海藥物所四年級(jí)博士研究生李平和陳筑熙,，是在蔣華良研究員,、高召兵副研究員和陽懷宇副研究員指導(dǎo)下完成。本研究工作得到科技部,、國(guó)家自然科學(xué)基金委,、上海市科委和NIH等項(xiàng)目的支持。（生物谷Bioon.com）
doi:10.1038/cr.2013.82
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The gating charge pathway of an epilepsy-associated potassium channel accommodates chemical ligands
Ping Li1,*, Zhuxi Chen2,*, Haiyan Xu1, Haifeng Sun2, Hao Li2, Hong Liu2, Huaiyu Yang2, Zhaobing Gao1, Hualiang Jiang2 and Min Li1,3
Voltage-gated potassium (Kv) channels derive their voltage sensitivity from movement of gating charges in voltage-sensor domains (VSDs). The gating charges translocate through a physical pathway in the VSD to open or close the channel. Previous studies showed that the gating charge pathways of Shaker and Kv1.2-2.1 chimeric channels are occluded, forming the structural basis for the focused electric field and gating charge transfer center. Here, we show that the gating charge pathway of the voltage-gated KCNQ2 potassium channel, activity reduction of which causes epilepsy, can accommodate various small molecule ligands. Combining mutagenesis, molecular simulation and electrophysiological recording, a binding model for the probe activator, ztz240, in the gating charge pathway was defined. This information was used to establish a docking-based virtual screening assay targeting the defined ligand-binding pocket. Nine activators with five new chemotypes were identified, and in vivo experiments showed that three ligands binding to the gating charge pathway exhibit significant anti-epilepsy activity. Identification of various novel activators by virtual screening targeting the pocket supports the presence of a ligand-binding site in the gating charge pathway. The capability of the gating charge pathway to accommodate small molecule ligands offers new insights into the gating charge pathway of the therapeutically relevant KCNQ2 channel.
