Proteolytic silencing of cancer targets using engineered ubiquitin ligases

描述（由申请人提供）：已经开发了多种技术，如基因靶向、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。总之，这些方法使得能够发现癌症中的新药物靶点，甚至能够治疗性地敲除致癌或促癌基因。然而，对于许多应用，可能期望靶向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传导途径中起着重要作用，并代表了寻求新的治疗癌症靶点的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，所有这些方法都靶向基因表达。因此，该提议寻求开发一种新的功能性蛋白质组学技术，该技术基于被称为“泛素”的抗体样E3泛素连接酶，其能够沉默靶蛋白及其后修饰的同种型。的 该方法的创新性在于产生泛体本身，其是抗体-酶嵌合体，其联合收割机E3泛蛋白连接酶（E3）的强蛋白水解活性与细胞内单链Fv（scFv）抗体的亲和力、特异性和模块性结合。所得到的遍在抗体能够募集细胞的强大的蛋白质降解系统，几乎任何感兴趣的蛋白质。由于可以产生泛抗体的scFv结构域以结合特定的蛋白质构象或翻译后修饰如糖基化或磷酸化，因此该技术具有沉默某些蛋白质的潜力。 异构体，同时保留其它异构体。长期目标是开发泛体作为一种创新的新型蛋白质沉默技术，用于癌症相关蛋白的功能分析以及癌症药物靶点的鉴定和药理学操作。为实现这一目标，提出了以下具体目标：首先，根据目标1，将针对非磷酸化的ERK 2是MAPK家族的成员，其成员众所周知调节几种生理和病理现象，包括炎症、凋亡性细胞死亡、致癌转化、肿瘤细胞侵袭和转移。其次，在目标2下，将检查抗ERK 2泛素异位表达对ERK 2信号传导以及哺乳动物细胞生长和增殖的影响。预计同种型特异性沉默将有助于揭示ERK在信号传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用于产生人工E3连接酶的简单且有效的工具，其使得能够：（i）分离不同功能的E3连接酶。 体细胞中细胞蛋白质的性质;（ii）评估特定细胞蛋白质是否是治疗干预的有效靶点;和（iii）消融细胞内蛋白质靶点作为某些人类恶性肿瘤如癌症的有效治疗策略。

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.