High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening
High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening
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DOI：
10.1074/jbc.m205932200
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发表时间：
2002-10-04
影响因子：
4.8
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Ma, TH;Vetrivel, L;Verkman, AS
囊性纤维化 (CF) 是由 CF 跨膜电导调节蛋白 (CFTR) 蛋白突变引起的，该蛋白会降低气道和其他上皮细胞中 cAMP 刺激的 Cl-电导。本研究的目的是确定新型有效的 CFTR 激活剂。晚上 10 点，在共同表达人 CFTR 和基于绿色荧光蛋白的 Cl 传感器的 Fisher 大鼠甲状腺上皮细胞中筛选了 60,000 种不同的药物样化合物以及低浓度的毛喉素 (0.5 μM)。初步筛选产生了 57 种强激活剂（比参考化合物芹菜素活性更高），其中大多数在化学结构上与已知的 CFTR 激活剂无关，以及 284 种较弱的激活剂。强激活剂的二次分析包括CFTR特异性、毛喉素需求、跨上皮短路电流、激活动力学、剂量反应、毒性和激活机制的分析。三种化合物（最有效的是二氢异喹啉）通过升高细胞 cAMP（可能是通过磷酸二酯酶抑制）来激活 CFTR。十四种化合物可激活 CFTR，但没有 cAMP 升高或磷酸酶抑制，表明 CFTR 存在直接相互作用。最有效的化合物具有四氢咔唑、羟基香豆素和噻唑烷核心结构。这些化合物快速诱导 CFTR Cl-电流（10 μM）。添加 10 分钟后，在 37°C 下生长的转染细胞中，没有任何化合物激活 DeltaPhe(508)-CFTR（DeltaPhe(508)-CFTR 被捕获在内质网中）。然而，在 27°C 下生长 48 小时校正运输后，四氢咔唑和 N-苯基三嗪衍生物强烈刺激 Cl- 电导，K-d < 1 μM。这里确定的新激活剂可能有助于定义 CFTR 激活的分子机制，并可作为 CF 药物开发的先导化合物。
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl- conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 pm together with a low concentration of forskolin (0.5 muM) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl- sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl- currents rapidly ( 10 muM). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degreesC (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degreesC, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl- conductance with K-d < 1 μM. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.