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

基本信息

批准号：
7340974
负责人：
DEAN R MADDEN
金额：
$ 41.83万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-15 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7340974
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 Condition Cyclization Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Defect Development Disruption Endocytosis Endopeptidases Epithelial Cells Exhibits Figs - dietary Fluorescence Polarization Gene Mutation Generations Goals Half-Life Hereditary Disease Human In Vitro Infection Knock-out Knowledge Lead Libraries Ligands Localized Lung Measurement Mediating Modification Mutagenesis Mutation PDZ protein Pathology Peptide Hydrolases Peptides Permeability Phage Display Pharmacologic Substance Pharmacological Treatment Phenylalanine Physiological Protein Binding Protein Overexpression 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 prevent programs protein aminoacid sequence protein function research study response scaffold skills small molecule small molecule libraries stereochemistry tool trafficking

项目摘要

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（申请人提供）：囊性纤维化（CF）是白种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，该突变表现出三大缺陷：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 AF508-CFTR 的细胞表面表达。该提议基于以下观察结果：(1)内源性 CAL 的 RNAi 敲除增加了极化 CF 患者气道上皮细胞系中 AF508-CFTR 的细胞表面表达和跨膜氯离子电流； (2) CAL 结合袋的局部、结构保守突变可阻断 CAL 介导的 CFTR 降解； (3)另一种 CFTR 结合蛋白 NHERF1 的过度表达也可以阻止降解。我们组建了一支高度协作的团队，拥有独特且互补的技能。我们的目标是有针对性地破坏关键的 CFTR 贩运相互作用及其功能表征。具体目标是：(1) 鉴定与 NHERF1 相比具有更高亲和力、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。将使用荧光偏振结合测定来筛选生物相容性小分子抑制剂。所有“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中 AF508-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重点关注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学测量来表征 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检测方法，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，以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南。除了定向合成之外，还将使用组合合成方法。优化的化合物将用于功能测定，并为潜在的药物开发提供基础。简单总结：在 CF 中，基因突变会阻止 CFTR 蛋白正常发挥作用，导致慢性肺部感染和死亡。我们寻找可以纠正这种功能缺陷的化学物质。