Boinot Clément, Jollivet Souchet Mathilde, Ferru-Clément Romain, Becq Frédéric
Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France.
Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France
J Pharmacol Exp Ther. 2014 Sep;350(3):624-34. doi: 10.1124/jpet.114.214890. Epub 2014 Jun 26.
The mutated protein F508del-cystic fibrosis transmembrane conductance regulator (CFTR) failed to traffic properly as a result of its retention in the endoplasmic reticulum and functions as a chloride (Cl(-)) channel with abnormal gating and endocytosis. Small chemicals (called correctors) individually restore F508del-CFTR trafficking and Cl(-) transport function, but recent findings indicate that synergistic pharmacology should be considered to address CFTR defects more clearly. We studied the function and maturation of F508del-CFTR expressed in HeLa cells using a combination of five correctors [miglustat, IsoLAB (1,4-dideoxy-2-hydroxymethyl-1,4-imino-l-threitol), Corr4a (N-[2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl]-benzamide), VX-809 [3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid], and suberoylamilide hydroxamic acid (SAHA)]. Using the whole-cell patch-clamp technique, the current density recorded in response to CFTR activators (forskolin + genistein) was significantly increased in the presence of the following combinations: VX-809 + IsoLAB; VX-809 + miglustat + SAHA; VX-809 + miglustat + IsoLAB; VX-809 + IsoLAB + SAHA; VX-809 + miglustat + IsoLAB + SAHA. These combinations restored the activity of F508del-CFTR but with a differential effect on the appearance of mature c-band of F508del-CFTR proteins. Focusing on the VX-809 + IsoLAB cocktail, we recorded a level of correction higher at 37°C versus room temperature, but without amelioration of the thermal instability of CFTR. The level of functional rescue with VX-809 + IsoLAB after 4 hours of incubation was maximal and similar to that obtained in optimal conditions of use for each compound (i.e., 24 hours for VX-809 + 4 hours for IsoLAB). Finally, we compared the stimulation of F508del-CFTR by forskolin or forskolin + VX-770 [N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide] with cells corrected by VX-809 + IsoLAB. Our results open new perspectives for the development of a synergistic polypharmacology to rescue F508del-CFTR and show the importance of temperature on the effect of correctors and on the level of correction, suggesting that optimized combination of correctors could lead to a better rescue of F508del-CFTR function.
突变蛋白F508del-囊性纤维化跨膜传导调节因子（CFTR）由于滞留在内质网而无法正常转运，并作为具有异常门控和内吞作用的氯离子（Cl(-)）通道发挥作用。小分子化合物（称为校正剂）可单独恢复F508del-CFTR的转运和Cl(-)转运功能，但最近的研究结果表明，应考虑采用协同药理学来更明确地解决CFTR缺陷问题。我们使用五种校正剂[米格鲁司他、IsoLAB（1,4-二脱氧-2-羟甲基-1,4-亚氨基-l-苏糖醇）、Corr4a（N-[2-(5-氯-2-甲氧基-苯氨基)-4'-甲基-[4,5']联噻唑-2'-基]-苯甲酰胺）、VX-809 [3-(6-(1-(2,2-二氟苯并[d][1,3]二氧杂环戊烯-5-基)环丙烷甲酰胺基)-3-甲基吡啶-2-基)苯甲酸]和辛二酰苯胺异羟肟酸（SAHA）]的组合，研究了在HeLa细胞中表达的F508del-CFTR的功能和成熟情况。使用全细胞膜片钳技术，在以下组合存在时，对CFTR激活剂（福斯可林 + 染料木黄酮）响应记录的电流密度显著增加：VX-809 + IsoLAB；VX-809 + 米格鲁司他 + SAHA；VX-809 + 米格鲁司他 + IsoLAB；VX-809 + IsoLAB + SAHA；VX-809 + 米格鲁司他 + IsoLAB + SAHA。这些组合恢复了F508del-CFTR的活性，但对F508del-CFTR蛋白成熟c带的出现有不同影响。聚焦于VX-809 + IsoLAB混合物，我们记录到37°C时的校正水平高于室温，但CFTR的热不稳定性没有改善。孵育4小时后，VX-809 + IsoLAB的功能挽救水平最高，与每种化合物在最佳使用条件下获得的水平相似（即VX-809为24小时 + IsoLAB为4小时）。最后，我们比较了福斯可林或福斯可林 + VX-770 [N-(2,4-二叔丁基-5-羟基苯基)-4-氧代-1,4-二氢喹啉-3-甲酰胺]对F508del-CFTR的刺激与经VX-809 + IsoLAB校正的细胞。我们的结果为开发协同多药理学以挽救F508del-CFTR开辟了新的前景，并表明温度对校正剂效果和校正水平的重要性，表明校正剂的优化组合可能导致更好地挽救F508del-CFTR功能。
