CFTR选择性抑制剂的发现及其在上皮液体分泌研究中的应用

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel, belongs to the adenine nucleotide-binding cassette (ABC) transporter family, and is the only anion channel of ABC superfamily. CFTR is extensively expressed on the luminal surface of serous epithelial cells most prominently in the intestines, airways, pancreas, bile ducts, epididymis, and conjunctiva. It has been confirmed that CFTR play a vital role in the absorption and secretion of electrolytes and fluid in submucosal glands. High potent CFTR specified inhibitor is still the most important tool in CFTR function studies so far. Due to low specificity, poor tissue distribution property and/or bad in vivo activity, none of the inhibitors identified so far was proved to fully meet the needs of CFTR functional studies in in vivo. Previously, we identified 42 active fractions that could inhibit CFTR chloride channel activities from 40,000 fraction libraries that generated from 500 most common used traditional Chinese herbs. Five active compounds (CFTRinh-N01~N05) from the positive fractions have been identified by further fractionation, activity analysis and structure determination. On the basis of our current work, we propose a four-year project to indentify 3~5 high potent CFTR blockerss that have potent inhibition efficiency in in vivo CFTR chloride channel activities. The proposed work includes: (1) Completion of fractionation, purification and structure determination of single compound inhibitors from the current 39 positive fractions with CFTR inhibition activities. (2) Completion of study of the specificity, mechasnism and molecular pharmacology of the active compounds by applying molecular biological and electrophysiological methods. (3) Completion primary in in vivo pharmacological studies of the active compounds on live tissue and mouse models. The CFTR inhibitors indentified in this project are important not only in CFTR channel gating mechanism studies, CFTR physiological and pathological functions investigations, but also important in pathological mechanism investigations and therapy of some CFTR-related diseases including polycystic kidney disease, secretory diarrhea, idiopathic chronic pancreatitis, and cystic fibrosis.

CFTR是cAMP依赖的氯离子通道，在哺乳动物所有与分泌和吸收有关的组织的上皮细胞中表达，在液体跨上皮转运中起关键作用。选择性通道抑制剂（blocker）是目前研究CFTR功能的最重要的药理学工具，现有抑制剂由于选择性或组织分布性差等因素使其应用受到极大限制。前期工作中，申请人成功建立了CFTR抑制剂高通量筛选细胞模型，并且从源自500种常用中草药的4万种中药组分中筛选出42个活性组分，目前已从中分离出5个活性化合物。本项目拟在此基础上，完成其余37个组分中活性化合物分离；应用细胞生物学和电生理手段对其选择性、作用机制和分子药理学特征进行研究；通过离体组织及体内上皮液体分泌研究确定其作为分子探针的有效性。目标是获得3～5种适合体内活性研究的CFTR选择性抑制剂。所获得的CFTR选择性抑制剂作为分子探针，将在深入研究CFTR在分泌性腹泻、多囊肾病、慢性胰腺炎等疾病的病理机制中发挥重要作用。

本项目按照计划书执行，完成了计划要点的全部内容。本项目中，我们利用功能测定细胞模型，对源自500余种中草药的天然小分子组分库（含40,000个组分）进行了筛选，利用活性引导的分离策略，从活性组分中分离获得了包括茋类、儿茶素类、四环二萜类、木脂素类、萘醌类等多个结构家族的23种CFTR氯离子通道抑制剂。我们利用Ussing-Chamber和内侧向外式（inside-out）膜片钳对活性化合物的电生理特征、选择性等基本药理学特性进行了分析，结果显示上述23种化合物均能以剂量依赖的方式抑制CFTR介导的氯离子电流，其中10种化合物表现出良好的体内活性。利用这些抑制剂分析了氯离子通道在肠道和呼吸道液体分泌中的作用，结合国际相关研究成果，基本明确了CFTR和CaCCs在肠道液体分泌和肠动力调节中的作用：跨上皮液体分泌主要是由肠上皮顶质膜上氯离子通道的主动转运驱动的；CFTR和一种分子身份未知的肠上皮CaCC是主要的氯离子通道，其中霍乱毒素和耐热性大肠杆菌内毒素等细菌毒素通过提高细胞内cAMP水平激活CFTR氯离子通道活性，轮状病毒NSP4蛋白和HIV蛋白酶抑制剂通过提高细胞内[Ca2+]激活CaCC；NSP4也能过激活肠道间质Cajal细胞上TMEM16A氯离子通道活性促进肠动力；抑制CFTR和CaCC能够通过抑制肠道液体分泌和促进肠吸收两个方面抑制腹泻的发生，而激活上述氯离子通道活性则能够逆转便秘。此外，在本项目中我们还发现几种天然小分子化合物对TMEM16A和肠上皮CaCC氯离子通道活性具有相反的调节作用，进一步证实了TMEM16A与肠上皮CaCC是分子身份不同的Ca2+激活氯离子通道。上述研究结果也解释了结肠上皮CaCC与气道上皮CaCC具有不同电导这一早期研究结果。本项目研究结果对于阐明CFTR的门控机制，揭示CFTR的生理病理功能，进一步阐明氯离子通道在慢性阻塞性肺病、慢性胰腺炎、多囊肾病以及囊性纤维化等CFTR和CaCCs相关疾病的发病机理和治疗中的作用奠定了基础。