Keeping CFTR in its Place: An Integrated Small-Molecule Approach

描述（由申请人提供）：囊性纤维化（CF）是白种人中最常见的遗传性疾病，由氯离子通道CFTR突变引起。90%的CF患者携带至少一个编码AF508-CFTR的突变拷贝，该突变表现出三个主要缺陷：内质网保留、通道功能受损和降解加速。我们假设选择性抑制cftr结合蛋白CAL会增加和稳定AF508-CFTR的细胞表面表达。该建议基于以下观察结果：(1)RNAi敲除内源性CAL增加了极化cf患者气道上皮细胞系中AF508-CFTR的细胞表面表达和跨膜氯电流；(2)局部的、结构保守的CAL结合口袋突变阻断了CAL介导的CFTR降解；(3)过表达另一种cftr结合蛋白NHERF1也可以阻断降解。我们已经组建了一支具有独特和互补技能的高度协作团队。我们的目标是有针对性的药物破坏关键的CFTR贩运相互作用及其功能表征。具体目的是：(1)与NHERF1相比，鉴定具有更高亲和力、生物利用度和选择性的CAL结合位点抑制剂。第一个选择性抑制剂已经被发现。基于肽的抑制剂将使用肽阵列和噬菌体展示技术进行检测。生物相容性小分子抑制剂将使用荧光偏振结合试验筛选。所有的“命中”都将通过二次生化筛选进行验证。(2)表征CAL抑制剂对极化气道上皮细胞AF508-CFTR细胞表面表达及氯离子通道活性的影响。对于肽抑制剂，递送试剂、细胞穿透肽序列或侧链环化将用于促进递送。我们的小分子筛选将专注于具有固有渗透性的化合物。交付后，我们将使用表面生物素化和电生理测量来表征AF508-CFTR救援和共同免疫沉淀实验，以量化CFTR:PDZ相互作用的破坏。可用的抑制剂将允许我们开始功能研究，并开发一种基于细胞的检测方法，用于新型抑制剂的三级化合物筛选。(3)对抑制剂先导化合物进行修饰，提高其亲和力和选择性；对多肽化合物进行修饰，优化其渗透性和蛋白水解稳定性。核磁共振将用于确定先导化合物的结合立体化学，作为化学修饰方法的指导。除了定向合成外，还将使用组合合成方法。优化后的化合物将用于功能分析，并将为潜在的药物开发提供基础。总结：在CF中，基因突变阻止CFTR蛋白正常工作，导致慢性肺部感染和死亡。我们寻找能够纠正这种功能缺陷的化学物质。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common genetic disease among Caucasians, and is caused by mutations of the chloride channel CFTR. 90% of CF patients carry at least one copy of the mutation encoding AF508-CFTR, which exhibits three major defects: ER retention, impaired channel function, and accelerated degradation. We hypothesize that selective inhibition of the CFTR-binding protein CAL will increase and stabilize cell-surface expression of AF508-CFTR. This proposal is based on the observations that (1) RNAi knock-down of endogenous CAL increases cell-surface expression of AF508-CFTR and transmembrane chloride currents in a polarized CF-patient airway epithelial cell line; (2) Localized, structurally conservative mutagenesis of the CAL binding pocket blocks CAL-mediated degradation of CFTR; and (3) Degradation can also be blocked by overexpression of another CFTR-binding protein, NHERF1. We have assembled a highly collaborative team with unique and complementary skills. Our goal is the targeted pharmacological disruption of a key CFTR trafficking interaction and its functional characterization. The specific aims are: (1) To identify inhibitors of the CAL binding site with improved affinity, bioavailability, and selectivity vs. NHERF1. A first selective inhibitor has already been found. Peptide-based inhibitors will be detected using peptide-array and phage-display technologies. Biocompatible small-molecule inhibitors will be screened using a fluorescence-polarization binding assay. All "hits" will be verified by secondary biochemical screens. (2) To characterize the effects of CAL inhibitors on cell-surface expression and chloride-channel activity of AF508-CFTR in polarized airway epithelial cells. For peptide inhibitors, delivery reagents, cell-penetrating peptide sequences, or side-chain cyclization will be used to facilitate delivery. Our small-molecule screens will focus on compounds with inherent permeability. Following delivery, we will use surface biotinylation and electrophysiological measurements to characterize AF508-CFTR rescue and co-immunoprecipitation experiments to quantify disruption of CFTR:PDZ interactions. The available inhibitor will allow us to begin functional studies and to develop a cell-based assay for tertiary compound screening of novel inhibitors. (3) To modify inhibitor lead compounds to enhance affinity and selectivity, and for peptide compounds, to optimize permeability and proteolytic stability. NMR will be used to determine binding stereochemistry of lead compounds, as a guide to chemical modification approaches. In addition to directed synthesis, combinatorial synthetic approaches will be used. Optimized compounds will be implemented in functional assays and will provide the basis for potential pharmaceutical development. Lay summary: In CF, genetic mutation prevents the CFTR protein from functioning correctly, leading to chronic lung infection and death. We seek chemicals that can correct this functional defect.