Proteolytic silencing of cancer targets using engineered ubiquitin ligases

描述(申请人提供)：已经开发了多种技术，如基因打靶、核酶、反义或RNA介导的干扰(RNAi)，用于沉默特定基因并评估其产物的生理功能。总而言之，这些方法正在使癌症新的药物靶点的发现，甚至是致癌或促癌基因的治疗性敲除成为可能。然而，对于许多应用来说，可能需要针对特定的蛋白质构象或翻译后修饰。特别是，蛋白质磷酸化在许多细胞内信号通路中发挥着重要作用，是寻找新的癌症治疗靶点的一个非常活跃的研究领域。不幸的是，使用上述方法不可能实现修饰特异性沉默，所有这些方法都是针对基因表达的。因此，这项提议试图开发一种新的功能蛋白质组学技术，该技术基于被称为泛体的抗体样泛素连接酶，能够沉默靶蛋白及其翻译后修饰的亚型。这个 这种方法的创新之处在于创造了泛体本身，这些泛体是抗体-酶嵌合体，结合了E3泛素连接酶(E3)强大的蛋白分解活性和细胞内单链抗体(ScFv)的亲和力、特异性和模块化。由此产生的泛体能够将细胞强大的蛋白质降解系统招募到几乎任何感兴趣的蛋白质中。由于泛体的scFv结构域可以被创建来结合特定的蛋白质构象或翻译后修饰，如糖基化或磷酸化，这项技术有可能沉默某些蛋白质 同型异构体，而放过其他人。其长期目标是开发泛体作为一种创新的新的蛋白质沉默技术，用于癌症相关蛋白的功能分析以及癌症药物靶标的识别和药理操作。为了实现这一目标，提出了以下具体目标：首先，在目标1下，将针对一个研究得很好的靶标ERK2的非磷酸化(非活性)和磷酸化(活性)亚型进行工程，ERK2是MAPK家族的成员，众所周知其成员调节多种生理和病理现象，包括炎症、凋亡细胞死亡、癌基因转化、肿瘤细胞侵袭和转移。其次，在目标2下，将研究抗ERK2泛体的异位表达对ERK2信号以及哺乳动物细胞的生长和增殖的影响。预计异构体特异性沉默将有助于揭示ERK在信号转导和细胞增殖中的作用的有价值的信息。更广泛地说，这种新的蛋白质敲除系统将提供一种简单而有效的工具来创建人工E3连接酶，从而能够：(I)解剖不同的功能 (2)评估特定的细胞蛋白是否是治疗干预的有效靶点；(3)消融细胞内蛋白靶点，以此作为治疗癌症等某些人类恶性肿瘤的有效治疗策略。

DESCRIPTION (provided by applicant): A variety of technologies such as gene targeting, ribozymes, antisense, or RNA-mediated interference (RNAi) have been developed for silencing of specific genes and assessing the physiological function of their products. Together, these approaches are enabling the discovery of novel drug targets in cancer and even the therapeutic knockdown of cancer-causing or cancer-promoting genes. However, for many applications it may be desirable to target specific protein conformations or post-translational modifications. In particular, protein phosphorylation plays a major role in numerous intracellular signaling pathways and represents a very active area of research in the quest for new therapeutic cancer targets. Unfortunately, modification-specific silencing is not possible using the above methods, all of which target gene expression. Therefore, this proposal seeks to develop a new functional proteomics technology based on antibody-like E3 ubiquitin ligase enzymes called "ubiquibodies" that are capable of silencing target proteins and their post-translationally modified isoforms. The innovativeness of this method lies in the creation of the ubiquibodies themselves, which are antibody-enzyme chimeras that combine the robust proteolytic activity of E3 ubiquitin ligases (E3s) with the affinity, specificity and modularity of intracellular single-chain Fv (scFv) antibodies. The resulting ubiquibodies are capable of recruiting the cell's robust protein degradation system to virtually any protein of interest. Since the scFv domain of a ubiquibody can be created to bind specific protein conformations or post-translational modifications such as glycosylation or phosphorylation, the technology has the potential for silencing of certain protein isoforms while sparing others. The long-term goal is to develop ubiquibodies as an innovative new protein silencing technology for functional analysis of cancer-associated proteins as well as for identification and pharmacological manipulation of cancer drug targets. Towards this goal, the following specific aims are proposed: first, under Aim 1, ubiquibodies will be engineered against the nonphosphorylated (inactive) and phosphorylated (active) isoforms of a well-studied target, ERK2, a member of the MAPK family whose members are well known to regulate several physiological and pathological phenomena including inflammation, apoptotic cell death, oncogenic transformation, tumor cell invasion, and metastasis. Second, under Aim 2, the consequences of ectopic expression of anti-ERK2 ubiquibodies will be examined with respect to ERK2 signaling as well as growth and proliferation of mammalian cells. It is anticipated that isoform-specific silencing will help to uncover valuable information about ERK's role in signaling and cell proliferation. More broadly, this new protein knockout system will provide a simple and efficient tool for creating artificial E3 ligases that enable: (i) dissection of diverse functional properties of cellular proteins in somatic cells; (ii) evaluation of whether specific cellular protins are valid targets for therapeutic intervention; and (iii) ablation of intracellular protein targetsas an effective therapeutic strategy for certain human malignancies such as cancer.