Development of selective substrate based inhibitors of ubiquitin isopeptidases

描述（由申请人提供）：FDA批准蛋白酶体抑制剂硼替佐米/VELCADE(r)用于治疗多发性骨髓瘤和其他血液系统恶性肿瘤，这表明泛素途径是治疗癌症和其他疾病的新药的有希望的来源。硼替佐米是一种蛋白酶体抑制剂，具有全球性作用，因此其使用受到严重毒性的限制。人们正在努力寻找比蛋白酶体更有选择性作用的泛素途径酶靶点，以开发具有更好的毒性特征和治疗指标的药物。这些酶包括泛素E1激活酶、E2结合酶、E3连接酶和去泛素化酶（DUBs）。dub作用于数量有限的靶蛋白，在生物化学和遗传上与包括癌症在内的各种疾病有关。这些酶是蛋白酶，被认为是可药物，各种制药和生物技术公司，包括Progenra已经确定了新的，选择性DUB抑制剂。尽管取得了这些进展，但尚无DUB抑制剂进入临床评估阶段。造成这种情况的部分原因可能是需要改进的筛选分析来确定临床前开发中抑制DUB作用于其原位底物的命中点。本申请中描述的项目是开发基于生理相关底物的检测方法，该方法比目前用于筛选DUB抑制剂的所有其他检测方法都有重大改进。该分析是新颖的，均匀的，符合hts，并利用特异性多泛素化DUB底物。将制备和纯化与治疗相关的DUB/泛素化底物对USP7/HDM2/p53（参与癌细胞增殖），配置测定方法，并进行抑制剂的中试筛选。这种新型的检测平台，在验证后，将扩展到包括更多的dub。拟议工作的最终商业目标是一种适用于HTS和可定制各种DUB/底物对的稳健、均质分析格式，从而使已知和新出现的DUB介导疾病的药物发现取得快速进展。

DESCRIPTION (provided by applicant): The FDA approval of the proteasome inhibitor bortezomib/VELCADE(r) for the treatment of multiple myeloma and other hematological malignancies demonstrates that the ubiquitin pathway is a promising source of new drugs for the treatment of cancer and perhaps other diseases. Bortezomib, a proteasome inhibitor, exerts a global effect and its use is thus limited by a serious toxicity profile. Efforts are underway to ind ubiquitin pathway enzyme targets acting more selectively than the proteasome with the aim of developing drugs with improved toxicity profiles and therapeutic indices. Among these enzymes are the ubiquitin E1 activating enzyme, E2 conjugating enzyme, E3 ligase and Deubiquitylases (DUBs). DUBs act on limited numbers of target proteins and are linked biochemically and genetically to various diseases, including cancer. These enzymes are proteases and are considered druggable, and various pharmaceutical and biotech companies, including Progenra have identified novel, selective DUB inhibitors. Despite this progress, no DUB inhibitors have progressed to clinical evaluation. Part of the reason for this situation may be that improved screening assays are needed to identify hits for preclinical development that inhibit the DUB acting upon its in situ substrate. The project described in this application is the development of physiologically relevant substrate- based assay that introduces a significant improvement over all of the other assays currently in use to screen for DUB inhibitors. The assay is novel, homogeneous, HTS-compliant, and utilizes specific polyubiquitylated DUB substrates. The therapeutically relevant DUB/ubiquitylated substrate pairs USP7/HDM2/p53 (involved in cancer cell proliferation) will be prepared and purified, the assay configured, and a pilot screen for inhibitors will be conducted. This novel assay platform, when validated, will be expanded to include additional DUBs. The ultimate commercial goal of the proposed work is a robust, homogenous assay format that is suitable for HTS and customizable to various DUB/substrate pairs, allowing rapid progress in drug discovery for known and emerging DUB-mediated diseases.