Applying Chemical Biology to Target Deubiquitinating Enzymes in Lung Cancer

泛素参与癌症发病机制的新证据重新引起了人们对设计的兴趣 操纵蛋白质泛素化的疗法。靶蛋白对赖氨酸的可逆泛素修饰 残基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、定位或蛋白质复合体。蛋白质泛素化的失调一直是 与癌症有关，从而提高了对这些信号蛋白的更好理解可以结合在一起的前景 用化学来创造新的疗法。许多关键途径已被证明受 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了。 影响蛋白质泛素化。战略包括瞄准E1-E2-E3级联的组件或瞄准 影响疾病过程的去泛素酶(DUB)。许多研究已经确定了潜在的 DUB抑制剂(DUBI)作为潜在的靶标。针对USP7的化学或遗传功能丧失研究， USP14、USP9X和UCHL5已被报道具有抗肿瘤特性。这个项目本来就是要开始的 通过重点开发化学生物学策略来提名蛋白质泛素化目标的研究 测定癌细胞和肿瘤组织中DUB的活性，并确定早期DUB抑制物的作用机制 化合物。我们建议使用一个化学生物学平台--基于活性的蛋白质图谱(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 针对酶的活性部位，探讨酶在生物中的作用状态 样本。这些基于活性的探针有两个关键元素：(I)与酶反应的基团和 共价标记它们的活性部位和(Ii)允许检测或浓缩 改良酶用于MS方法检测。使用Dub ABPP探针的初步数据显示 利用这项技术分析DUB的可行性以及DUBI对肺癌细胞的影响。目标1将 用ABPP鉴定肺癌细胞系和肿瘤中的Dub蛋白。DUB探头将用于 DUB信号在多种组织学亚型肺癌中的特征，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已建立的细胞系 将被配置和注释到已建立的DUB信号通路。目标2将审问职能部门 用ABPP鉴定已发现的DUB蛋白对识别重要的肺调节靶点的意义 癌症的生存能力。第一种方法是候选方法，其中使用ABPP确定的DUB将是 使用RNA干扰和小分子干扰技术研究它们在促进肺癌生长和存活方面的作用 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性来识别“驾驶员”副本 利用ABPP进行目标识别的杜比表型筛选。DUBI诱导的细胞活力变化 肺癌细胞株将与竞争性配对图谱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.