Applying Chemical Biology to Target Deubiquitinating Enzymes in Lung Cancer

应用化学生物学靶向肺癌中的去泛素化酶

• 批准号: 9375662
• 负责人: ERIC B. HAURA
• 金额: $ 22.45万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-03 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375662
• 关键词: Active Sites; Adenocarcinoma; Affect; Biological; Biological Assay; Biology; Cancer cell line; Cell Line; Cell Survival; Cells; Chemicals; Chemistry; Computer software; Data; Detection; Deubiquitinating Enzyme; Disease; Drug Targeting; Elements; Enzyme Inhibitor Drugs; Enzymes; Epidermal Growth Factor Receptor; Excision; Family; Genetic; Goals; Growth; Histologic; Histology; Individual; KRAS2 gene; Label; Laboratories; Link; Lysine; Malignant Neoplasms; Malignant neoplasm of lung; Manipulative Therapies; Mass Spectrum Analysis; Measures; Mediating; Medicine; Methodology; Methods; Modeling; Monitor; Non-Small-Cell Lung Carcinoma; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Peptides; Performance; Phenotype; Post-Translational Protein Processing; Process; Property; Protein Kinase; Proteins; Proteomics; RNA Interference; Reporter; Reporting; Resistance; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Squamous Cell; Technology; Testing; Therapeutic Intervention; Time; Tumor Cell Line; Tumor Tissue; Ubiquitin; Ubiquitination; activity-based protein profiling; base; cancer cell; cancer survival; enzyme activity; established cell line; high reward; high risk; inhibitor/antagonist; interest; loss of function; lung small cell carcinoma; mass spectrometer; multicatalytic endopeptidase complex; mutant; novel therapeutics; protein complex; small molecule; small molecule inhibitor; targeted treatment; therapy design; tool; tumor

Emerging evidence implicating ubiquitination in the pathogenesis of cancer has renewed interest in designed therapies that manipulate protein ubiquitination. Reversible ubiquitin modification of target proteins on lysine residues results in major changes in signaling proteins, either through degradation through the proteasome, or by altering the activity, localization, or protein complexes. Dysregulation of protein ubiquitination has been linked to cancer thus raising the promise that better understanding of these signaling proteins can be combined with chemistry to create new therapeutics. A number of key pathways have been shown to be regulated by protein ubiquitination. Based on these observations, there is increased enthusiasm for targeting proteins affecting protein ubiquitination. Strategies include targeting components of the E1-E2-E3 cascade or targeting deubiquitinating enzymes (DUB) that affect disease processes. A number of studies have identified potential DUB inhibitors (DUBi) as potential targets. Chemical or genetic loss of function studies targeting USP7, USP14, USP9X, and UCHL5 have been reported with anti-tumor properties. This project is meant to begin studies nominating protein ubiquitination targets by focusing on developing chemical biology strategies to profile DUB activity in cancer cells and tumor tissues, and define mechanism of action of early DUB inhibitor compounds. We propose to use a chemical biology platform, activity-based protein profiling (ABPP), to study the activity of DUB in lung cancer and nominate new potential targets. ABPP uses chemical probes that are directed against the active sites of enzymes to interrogate the functional state of enzymes in biological samples. These activity-based probes have two critical elements: (i) a group that reacts with enzymes and covalently labels their active sites and (ii) a reporter tag that allows for the detection or enrichment of the modified enzymes for detection using MS approaches. Preliminary data with DUB ABPP probes demonstrates feasibility of using this technology to profile DUB and the effects of DUBi in lung cancer cells. Aim 1 will characterize DUB proteins in lung cancer cell lines and tumors using ABPP. DUB probes will be used to characterize DUB signaling in multiple histological subtypes of lung cancer, including adenocarcinoma, squamous cell, and small cell. Tumor tissues taken at the time of surgical resection and established cell lines will be profiled and annotated to established DUB signaling pathways. Aim 2 will interrogate the functional significance of discovered DUB proteins identified using ABPP to identify important targets that mediate lung cancer viability. The first approach is a candidate approach, where DUB identified using ABPP will be examined for their role in promoting lung cancer growth and survival using RNA interference and small molecule inhibitors in cell line models. The second approach is to identify “driver” DUB using a target-agnostic phenotypic screen of DUBi with target identification using ABPP. Changes in cell viability induced by DUBi in lung cancer cell lines will be used alongside competitive DUB profiling to nominate & validate targets.

新的证据表明泛素化在癌症发病机制中的作用重新引起了人们对设计的兴趣 操纵蛋白质泛素化的疗法。靶蛋白赖氨酸可逆泛素修饰 残留物导致信号蛋白发生重大变化，要么通过蛋白酶体降解，要么 通过改变活性、定位或蛋白质复合物。蛋白质泛素化失调 与癌症相关，因此有望更好地理解这些信号蛋白 与化学一起创造新的疗法。许多关键途径已被证明受到调节 蛋白质泛素化。基于这些观察，人们对靶向蛋白质的热情日益高涨 影响蛋白质泛素化。策略包括E1-E2-E3级联的靶向成分或靶向 影响疾病过程的去泛素化酶（DUB）。许多研究已经确定了潜在的 DUB 抑制剂 (DUBi) 作为潜在靶点。针对 USP7 的化学或遗传功能丧失研究， 据报道，USP14、USP9X 和 UCHL5 具有抗肿瘤特性。该项目即将开始 通过专注于开发化学生物学策略来研究提名蛋白质泛素化靶标 分析癌细胞和肿瘤组织中的 DUB 活性，并确定早期 DUB 抑制剂的作用机制 化合物。我们建议使用化学生物学平台，基于活性的蛋白质分析（ABPP）来研究 DUB 在肺癌中的活性并提名新的潜在靶点。 ABPP 使用的化学探针是 针对酶的活性位点来询问生物中酶的功能状态 样品。这些基于活性的探针有两个关键要素：（i）与酶反应的基团和 共价标记其活性位点和（ii）报告标签，允许检测或富集 使用 MS 方法进行检测的修饰酶。 DUB ABPP 探针的初步数据表明 使用该技术来分析 DUB 的可行性以及 DUBi 对肺癌细胞的影响。目标1将 使用 ABPP 表征肺癌细胞系和肿瘤中的 DUB 蛋白。 DUB 探针将用于 表征肺癌多种组织学亚型（包括腺癌）中的 DUB 信号传导， 鳞状细胞、小细胞。手术切除时采集的肿瘤组织和建立的细胞系 将针对已建立的 DUB 信号通路进行分析和注释。目标 2 将询问功能 使用 ABPP 鉴定发现的 DUB 蛋白对于识别介导肺的重要靶标的重要性 癌症生存能力。第一种方法是候选方法，其中使用 ABPP 识别的 DUB 将是 使用 RNA 干扰和小分子检测它们在促进肺癌生长和生存中的作用 细胞系模型中的分子抑制剂。第二种方法是使用与目标无关的方法来识别“驱动程序”DUB 使用 ABPP 对 DUBi 进行表型筛选并进行目标鉴定。 DUBi 诱导的细胞活力变化 肺癌细胞系将与竞争性 DUB 分析一起使用来提名和验证靶点。