Physiology Department, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
Mol Pharmacol. 2010 Jun;77(6):922-30. doi: 10.1124/mol.109.062679. Epub 2010 Mar 3.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation Delta Phe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a Delta Phe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 microM). Glafenine increased the surface expression of Delta Phe508-CFTR in baby hamster kidney (BHK) cells to approximately 40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o(-) monolayers and intestines isolated from Delta Phe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (I(sc)) response to forskolin + genistein, and this effect was abolished by 10 microM CFTR(inh)172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.
囊性纤维化(CF)是由 CF 跨膜电导调节因子(CFTR)基因突变引起的,该基因编码在上皮细胞中表达的 cAMP 激活阴离子通道。最常见的突变 Delta Phe508 导致蛋白质错误折叠、内质网滞留和降解。一种有前途的治疗方法是鉴定已为其他适应症开发但也能纠正 CFTR 运输缺陷的药物,从而利用它们在人类中的已知安全性和生物利用度,并减少临床开发所需的时间。我们使用 Delta Phe508-CFTR 运输测定法筛选了具有已知安全性和生物利用度的已批准、上市和非专利药物。在确认的命中药物中,有一个是 glafenine,一种具有镇痛特性的邻氨基苯甲酸衍生物。其纠正 CFTR 错误加工的能力通过使用在血浆中达到临床浓度(10 μM)的体外和体内研究得到证实。Glafenine 将 Delta Phe508-CFTR 在婴儿仓鼠肾(BHK)细胞中的表面表达增加到约野生型 CFTR 的 40%,与已知的 CFTR 校正剂 4-环己氧基-2-{1-[4-(4-甲氧基苯磺酰基)-哌嗪-1-基]-乙基}-喹唑啉(VRT-325)相当。Western blot 中成熟 CFTR 的出现以及未极化 BHK 和人胚肾细胞中两个卤化物通透性测定证实了部分纠正。用 glafenine 孵育极化 CFBE41o(-)单层和从 Delta Phe508-CFTR 小鼠(离体处理)分离的肠,增加了 forskolin + genistein 对短电路电流(I(sc))的反应,而 10 μM CFTR(inh)172 则消除了这种作用。体内用 glafenine 治疗也部分恢复了总唾液分泌。我们得出结论,发现 glafenine 作为 CFTR 校正剂验证了研究现有药物治疗 CF 的方法,尽管可能需要局部给药或进一步的药物化学来减少副作用。