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野生型和突变CFTR氯离子通道天然小分子激活剂的发现与分子药理学研究
Discovery and Pharmacological Characterization of Natural Small Molecule Activators of Wildtype and Mutant CFTR Chloride Channels
【作者】 林森；
【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2008， 硕士
【摘要】 本文工作的目的是对中药单体化合物荷叶碱和姜黄素的CFTR氯离子通道激活作用进行系统的分子药理学研究,以评价其在治疗CF疾病中的可能性和策略。CFTR由于其突变能导致致命性遗传疾病CF而备受关注,人们对其结构、功能进行大量研究取得了很大成果。目前,寻找能够激活野生型和突变型CFTR功能的小分子调节剂是CFTR有关研究的重点之一。我们实验组在前期实验中利用稳定表达人CFTR和对卤族元素碘离子高度敏感的荧光绿蛋白突变体EYFP-H148Q的Fischer大鼠甲状腺上皮细胞为筛选模型,在中药单体化合物库中发现了大量野生型和突变型CFTR Cl~-通道激活剂。在此基础上我们选择了荷叶碱和姜黄素,利用荧光细胞功能测定模型、短路电流测定技术以及细胞膜片钳技术等手段对二者的CFTR氯离子通道激活作用进行系统的分子药理学研究。结果表明,生物碱类化合物荷叶碱对野生型和ΔF508突变型CFTR Cl~-通道具有激活作用,而对G551D突变型CFTR Cl~-通道无激活作用。黄酮类化合物姜黄素不能纠正ΔF508-CFTR蛋白胞内转运的障碍,但却具有纠正其通道开放障碍的功能;而且姜黄素对G551D突变型CFTR Cl~-通道也有激活作用。上述两种化合物对野生型和突变型CFTR Cl~-通道的激活作用具有作用迅速、可逆、剂量依赖的特点,上述活性依赖于细胞内cAMP水平而不提高细胞内cAMP水平。机制分析结果显示它们可能是通过与CFTR直接结合而发挥作用的荷叶碱与姜黄素具有多方面药理作用,是对CFTR具有激活作用的天然化合物之一,将在阐明CFTR活性机制及作为先导化合物开发与CFTR有关的疾病治疗药物等方面具有重要用途。
【Abstract】 Content:The purpose of this study was to investigate the molecular pharmacological mechanism of two natural compounds,nuciferine and curcumin,on CFTR chloride channel in order to evaluate possibility and strategy of their potential use in CF therapy.Mutations of CFTR may lead to the lethal genetic disease Cystic fibrosis (CF),so many attentions have been paid to the CFTR study.Great endeavors have been paid to the study CFTR,and great progress has been achieved in small molecule CFTR regulator identification.Previously,our lab identified a group of CFTR(both wild-type and mutant forms) activators from Chinese herbs by using a cell-based fluorescence assay.Based on this,in this paper we systematically investigated effect of nuciferine and curcumin on CFTR chloride channel activity by using the fluorescence assay,Ussing chanmber short-circuit current and patch-clamp technique.We demonstrated that nuciferine activated both wild-type and AF508 mutant CFTR Cl-channel activity,but had no effect on G551D mutant CFTR;curcumin corrected both AF508 and G551D mutant CFTR channel gating defect,but did not correct AF508 mutant CFTR processing defect.Activation of CFTR by these potentiators is rapid,reversible,dose-dependent and cAMP-dependent,but does not elevate cellular cAMP level.Mechanism analyses suggest that they work by a direct binding to CFTR molecule way.As natural CFTR activators,nuciferine and curcumin may be useful for probing CFTR channel gating mechanisms and as a lead compound to develop pharmacological therapy of CFTR-related disease.