Identification of natural coumarin compounds that rescue defective ΔF508-CFTR chloride channel gating
Identification of natural coumarin compounds that rescue defective ΔF508-CFTR chloride channel gating
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DOI：
10.1111/j.1440-1681.2008.04943.x
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发表时间：
2008-08-01
影响因子：
2.9
通讯作者：
Ma, Tong-Hui
中科院分区：
文献类型：
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作者：
Xu, Li-Na;Na, Wan-Li;Ma, Tong-Hui
1.囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的508位苯丙氨酸（Delta F508）的缺失是引起囊性纤维化（CF）的最常见突变。Delta F508-CFTR突变引起的CF的有效药物治疗需要拯救细胞内加工和通道门控缺陷。我们通过从中草药中筛选386个单一天然化合物，鉴定了一类可以纠正缺陷的Delta F508-CFTR氯离子通道门控的天然香豆素化合物。在共表达Delta F508-CFTR和碘敏感性荧光指示剂（YFP-H148 Q/I152 L）的Fischer大鼠甲状腺上皮细胞中用碘流入测定进行筛选。通过荧光碘离子内流试验证明了五种香豆素化合物对缺陷性Delta F508-CFTR氯离子通道门控的剂量依赖性增强作用，并通过Ussing室短路电流试验证实。激活被特异性CFTR抑制剂CFTRinh-172完全消除。两种有效的化合物，即欧前胡素和蛇床子素，具有约10 mmol/L的活化Kd值，如通过短路电流测定所确定的。活性香豆素化合物不升高细胞内cAMP水平。通过香豆素化合物激活Delta F508-CFTR需要cAMP激动剂，表明与突变CFTR分子的直接相互作用。动力学分析表明，香豆素类化合物对Delta F508-CFTR的活化迅速，半数最大活化时间< 5 min，洗脱后45 min，所有5种活性化合物的活化作用均完全逆转.总之，天然香豆素Delta F508-CFTR激活剂可能代表一类新的天然先导化合物，用于开发Delta F508突变引起的CF的药理学疗法。
1. Deletion of phenylalanine at position 508 (Delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the Delta F508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects.2. We identified a class of natural coumarin compounds that can correct the defective Delta F508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing Delta F508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L).3. Dose-dependent potentiation of defective Delta F508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTRinh-172. Two potent compounds, namely imperatorin and osthole, have activation K-d values of approximately 10 mmol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of Delta F508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of Delta F508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout.4. In conclusion, the natural coumarin Delta F508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the Delta F508 mutation.