Applying Chemical Biology to Target Deubiquitinating Enzymes in Lung Cancer

新出现的证据表明，泛素化在癌症发病机制中的作用重新引起了人们对设计 操纵蛋白质泛素化的疗法。靶蛋白在赖氨酸上的可逆泛素修饰 残基导致信号蛋白发生重大变化，或者通过蛋白酶体降解，或者 通过改变活性、定位或蛋白质复合物。蛋白质泛素化的失调已经被 与癌症有关，因此提高了对这些信号蛋白的更好理解的希望， 来创造新的疗法。许多关键途径已被证明受到以下因素的调节 蛋白质泛素化基于这些观察，人们对靶向蛋白质的热情增加 影响蛋白质泛素化。策略包括靶向E1-E2-E3级联反应的组分或靶向 去泛素化酶（DUB）影响疾病过程。一些研究已经发现了潜在的 DUB抑制剂（DUBi）作为潜在的目标。针对USP 7的化学或遗传功能丧失研究， USP 14、USP 9 X和UCHL 5已被报道具有抗肿瘤特性。这个项目是为了开始 研究提名蛋白质泛素化的目标，重点是发展化学生物学策略， 描述DUB在癌细胞和肿瘤组织中的活性，并确定早期DUB抑制剂的作用机制 化合物.我们建议使用一个化学生物学平台，基于活性的蛋白质谱（ABPP），研究 DUB在肺癌中活性并提名新的潜在靶点。ABPP使用化学探针， 针对酶的活性位点来询问生物体内酶的功能状态， 样品这些基于活性的探针具有两个关键要素：（i）与酶反应的基团， 共价标记它们的活性位点，和（ii）允许检测或富集它们的活性位点的报告物标签。 修饰的酶用于使用MS方法检测。DUB ABPP探针的初步数据表明， 使用该技术分析DUB的可行性以及DUBi在肺癌细胞中的作用。目标1将 使用ABPP表征肺癌细胞系和肿瘤中的DUB蛋白。DUB探头将用于 表征肺癌的多种组织学亚型中的DUB信号传导，包括腺癌， 鳞状细胞和小细胞。手术切除时采集的肿瘤组织和建立的细胞系 将被分析并注释到已建立的DUB信号通路。目标2将询问功能 使用ABPP鉴定发现的DUB蛋白对鉴定介导肺损伤的重要靶点的意义 癌症生存能力。第一种方法是候选方法，其中将使用ABPP识别DUB。 研究了它们在促进肺癌生长和生存中的作用，使用RNA干扰和小 细胞系模型中的分子抑制剂。第二种方法是使用目标不可知的 使用ABPP进行DUBi的表型筛选和靶标鉴定。DUBi诱导的细胞活力变化 肺癌细胞系将与竞争性DUB分析一起用于提名和验证靶点。

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.