Small-Molecule Targeting of CFTR:PDZ Trafficking Interactions

囊性纤维化（CF）是白种人中最常见的致命性遗传疾病，由编码氯离子通道CFTR的基因突变引起。90%的CF患者携带至少一个编码F508-CFTR的突变拷贝，该突变表现出三个主要缺陷：生物合成效率低下、通道功能受损和降解加速。虽然前两种已经成为广泛的药理学研究的焦点，但第三种缺陷的特征却很差。我们现在已经证明cftr相关配体CAL参与调节F508-CFTR的成熟后稳定性。CAL含有PDZ （PSD-95/DLG/ZO-1）蛋白-蛋白相互作用结构域，该结构域与CFTR c端结合，是CAL介导的CFTR降解所必需的。相应的，CAL抑制增加了极化人气道上皮细胞中F508-CFTR的顶膜半衰期和净功能表达水平。鉴于缺乏CAL PDZ结构域的小分子探针，我们设计了选择性抑制CAL结合的肽，用于这些原理验证研究。然而，对CAL作用的机制理解将需要解剖CAL和其他PDZ蛋白在调节F508-CFTR稳定性的内吞途径中的竞争作用。能够解决这个问题的抑制剂应该避免CAL特异性RNAi所需的长孵育时间，同时也避免与肽递送和稳定性相关的体内功效问题。为了鉴定类似药物的小分子抑制剂，我们现在建立了基于正交肽的FRET和alphasgreen报告基因测定法。在这里，我们建议结合这些分析来开发一个平台，以高通量筛选模式识别选择性CAL抑制剂，并使用5600种生物活性化合物的样品阵列来验证该平台。通过这些技术在单点测定中鉴定的化合物将进行剂量-反应分析。剂量反应点相互作用的有效性和位置将使用已建立的核磁共振分析来确定，既可以作为三级筛选，也可以在需要时促进组合化学方法。确认的命中将被反筛选，因为缺乏对NHERF1和NHERF2 PDZ结构域的抑制，其与CFTR的相互作用稳定了细胞表面功能性氯离子通道的表达，并进行生物相容性测试。我们的目标是配置一个强大的方案来筛选CAL抑制剂的大化合物文库，用于我们的极化人气道上皮细胞系统的下游测试。

Principal Investigator/Program Director (Last, First, Middle): MADDEN, Dean R. Cystic fibrosis (CF) is the most common fatal genetic disorder among Caucasians and is caused by mutations in the gene encoding the chloride channel CFTR. Ninety percent of CF patients carry at least one copy of the mutation encoding F508-CFTR, which exhibits three major defects: inefficient biosynthesis, impaired channel function, and accelerated degradation. While the first two have been the focus of extensive pharmacological investigation, the third defect is poorly characterized. We have now shown that the CFTR-Associated Ligand CAL is implicated in regulating the post-maturational stability of F508-CFTR. CAL contains a PDZ (PSD-95/DLG/ZO-1) protein-protein interaction domain that binds to the CFTR C-terminus and is required for CAL-mediated degradation of CFTR. Correspondingly, CAL inhibition increases the apical membrane half-life and net functional expression level of F508-CFTR in polarized human airway epithelial cells. Given a lack of small-molecule probes for the CAL PDZ domain, we engineered peptides that selectively inhibit CAL binding for these proof-of-principle studies. However, a mechanistic understanding of CAL's effect will require dissection of the competing roles of CAL and other PDZ proteins in the endocytic pathways that modulate F508-CFTR stability. Inhibitors that would allow us to address this question should avoid the long incubation times required for CAL- specific RNAi, and also avoid the in vivo efficacy issues associated with peptide delivery and stability. To identify drug-like small-molecule inhibitors, we have now established orthogonal peptide-based FRET and AlphaScreen reporter assays. Here, we propose to combine these assays to develop a platform for identifying selective CAL inhibitors in high-throughput screening mode and to validate the platform using a sample array of 5600 bioactive compounds. Compounds identified by these techniques in single-point assays will be subjected to dose-response analysis. The validity and site(s) of interaction of dose-responding hits will be determined using an established NMR assay, both as a tertiary screen and to facilitate combinatorial chemistry approaches if needed. Confirmed hits will be counterscreened for lack of inhibition of the NHERF1 and NHERF2 PDZ domains, whose interaction with CFTR stabilizes cell-surface expression of functional chloride channels, and tested for biocompatibility. Our goal is to configure a robust protocol to screen large compound libraries for CAL inhibitors for downstream testing in our polarized human airway epithelial-cell system.