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

描述(申请人提供)：囊性纤维化(CF)是高加索人中最常见的遗传病，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，表现出三个主要缺陷：内质网滞留、通道功能受损和加速降解。我们假设选择性地抑制CFTR结合蛋白CAL将增加和稳定AF508-CFTR在细胞表面的表达。这一建议是基于以下观察结果：(1)内源性CAL的RNAi下调增加了极化的CF患者呼吸道上皮细胞系AF508-CFTR的细胞表面表达和跨膜氯电流；(2)CAL结合口袋的局部、结构保守的突变阻断了CAL介导的CFTR的降解；(3)另一种CFTR结合蛋白NHERF1的过表达也可以阻止CFTR的降解。我们组建了一支高度协作的团队，拥有独特和互补的技能。我们的目标是有针对性地从药理上破坏一个关键的CFTR贩运相互作用及其功能特征。具体目标是：(1)确定与NHERF1相比，具有更好的亲和力、生物利用度和选择性的CAL结合位点的抑制剂。已经发现了第一种选择性抑制剂。基于多肽的抑制剂将使用多肽阵列和噬菌体展示技术进行检测。生物相容的小分子抑制剂将使用荧光偏振结合试验进行筛选。所有的“命中”都将通过二级生化筛查进行核实。(2)研究CAL抑制剂对极化气道上皮细胞表面AF508-CFTR表达和氯通道活性的影响。对于多肽抑制剂，将使用递送试剂、细胞穿透性多肽序列或侧链环化来促进递送。我们的小分子筛选将重点放在具有固有渗透性的化合物上。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救援和免疫共沉淀实验，以量化CFTR：PDZ相互作用的破坏。可用的抑制剂将使我们能够开始功能研究，并开发一种基于细胞的分析方法，用于新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，以提高亲和力和选择性，对于多肽化合物，则优化通透性和蛋白降解稳定性。核磁共振将被用来确定铅化合物的结合立体化学，作为化学修饰方法的指南。除了定向合成外，还将使用组合合成方法。优化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：在慢性萎缩性胃炎中，基因突变阻止了CFTR蛋白的正常功能，导致慢性肺部感染和死亡。我们正在寻找能够纠正这一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.