Proteolytic silencing of cancer targets using engineered ubiquitin ligases

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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.