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

保持 CFTR 的位置：一种集成的小分子方法

• 批准号: 8009409
• 负责人: DEAN R MADDEN
• 金额: $ 41.73万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-15 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009409
• 关键词: Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; Biocompatible; Biological; Biological Assay; Biological Availability; Biotinylation; Caucasians; Caucasoid Race; Cell Line; Cell membrane; Cell surface; Cells; Cessation of life; Chemicals; Chloride Channels; Chloride Ion; Chlorides; Chronic; Co-Immunoprecipitations; Combinatorial Synthesis; Complex; Cyclization; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Development; Endocytosis; Epithelial Cells; Exhibits; Fluorescence Polarization; Gene Mutation; Generations; Goals; Half-Life; Hereditary Disease; Human; In Vitro; Infection; Knock-out; Knowledge; Lead; Libraries; Ligands; Lung; Measurement; Mediating; Modification; Mutagenesis; Mutation; PDZ protein; Pathology; Peptide Hydrolases; Peptides; Permeability; Phage Display; Pharmacologic Substance; Pharmacological Treatment; Phenylalanine; Physiological; Protein Binding; Proteins; RNA Interference; Reagent; Research; Research Personnel; Resistance; Screening procedure; Side; Small Interfering RNA; Structure; Surface; Surveys; System; Technology; Testing; Therapeutic Intervention; Translational Research; Work; apical membrane; base; combinatorial; cystic fibrosis patients; design; disease-causing mutation; improved; in vivo; inhibitor/antagonist; knock-down; mutant; novel; overexpression; prevent; programs; protein aminoacid sequence; protein function; research study; response; scaffold; skills; small molecule; small molecule libraries; stereochemistry; tool; trafficking

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.

描述（由申请人提供）：囊性纤维化（CF）是高加索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患者携带至少一个拷贝的编码AF 508-CFTR的突变，其表现出三个主要缺陷：ER滞留、通道功能受损和加速降解。我们假设选择性抑制CFTR结合蛋白CAL将增加并稳定AF 508-CFTR的细胞表面表达。该提议基于以下观察：（1）内源性CAL的RNAi敲低增加极化CF患者气道上皮细胞系中AF 508-CFTR的细胞表面表达和跨膜氯电流;（2）CAL结合口袋的局部、结构保守的诱变阻断CAL介导的CFTR降解;和（3）降解也可以通过另一种CFTR结合蛋白NHERF 1的过表达来阻断。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的药理学破坏一个关键的CFTR运输相互作用及其功能表征。具体目的是：（1）鉴定CAL结合位点的抑制剂，其与NHERF 1相比具有改善的亲和力、生物利用度和选择性。第一个选择性抑制剂已经被发现，基于肽的抑制剂将利用肽阵列和噬菌体展示技术检测。将使用荧光偏振结合试验筛选生物相容性小分子抑制剂。所有“命中”将通过二级生化筛选进行验证。(2)表征CAL抑制剂对极化气道上皮细胞中AF 508-CFTR的细胞表面表达和氯离子通道活性的影响。对于肽抑制剂，将使用递送试剂、细胞穿透肽序列或侧链环化来促进递送。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AF 508-CFTR拯救和免疫共沉淀实验，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测定方法，用于新型抑制剂的三级化合物筛选。(3)修饰抑制剂先导化合物以增强亲和力和选择性，对于肽化合物，优化渗透性和蛋白水解稳定性。NMR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。除了定向合成之外，还将使用组合合成方法。优化的化合物将在功能测定中实施，并将为潜在的药物开发提供基础。在CF中，基因突变会阻止CFTR蛋白正常发挥功能，导致慢性肺部感染和死亡。我们正在寻找可以纠正这种功能缺陷的化学物质。