HTS for Rpn11 chemical probes

描述（由申请人提供）：蛋白酶体是主要负责真核细胞的细胞质和细胞核内蛋白质周转的酶。 指定用于蛋白酶体的底物首先与泛素链缀合，泛素链与蛋白酶体上的受体结合，从而束缚底物，使得其可以移位到蛋白酶体的内室中，在那里其被降解。 许多临床研究已经证实，阻断蛋白酶体内腔中的蛋白降解活性位点的小分子可以阻止人类癌症的进展，包括多发性骨髓瘤和套细胞淋巴瘤。 在以前的工作中，我们发现，Rpn 11亚基的蛋白酶体含有一个新的锌金属蛋白酶活性位点，去除泛素链的蛋白酶体底物。 这种由Rpn 11催化的“去泛素化”对于蛋白酶体的蛋白降解活性是必不可少的，大概是因为庞大的泛素链阻止底物插入蛋白酶体的内室。 因此，Rpn 11金属蛋白酶代表了一种新的蛋白酶体活性，可以潜在地被小分子靶向以阻断蛋白酶体底物的降解并阻止人类肿瘤的生长。 本申请的目标是使用该检测方法从位于Conrad Prebys化学基因组学中心的MLPCN中筛选大型化合物库（约330，000种化合物），以鉴定Rpn 11抑制剂，并在二级和三级检测中对这些抑制剂进行分类，这些检测旨在优先考虑强效、选择性、竞争性、可逆和细胞可渗透的命中。 我们的策略是基于一个基板，包括四个串联的泛素融合到一个肽带有一个独特的半胱氨酸，可以用荧光团标记。 Rpn 11在第四个泛素和肽之间的连接处切割该底物释放低分子量荧光肽，导致荧光团的大去极化。 通过测量在26 S蛋白酶体加上各种化合物的存在下底物的荧光偏振，我们应该能够识别那些化合物，称为命中，阻断Rpn 11依赖性释放的荧光肽产物。 随后通过二级和三级试验的命中将为后续SAR研究提供信息，以改善化合物效力、选择性和细胞渗透性。 这些抑制剂将用于区分蛋白酶体的泛素依赖性和泛素非依赖性底物，并测试抑制Rpn 11是否是癌症化疗的可行策略。 公共卫生相关性：蛋白酶体是新型药物--“蛋白酶体抑制剂”--的靶点，用于治疗血液恶性肿瘤、多发性骨髓瘤和套细胞淋巴瘤。 几年前，我们发现了蛋白酶体的一种新活性，我们将其命名为Rpn 11异肽酶。 用药物靶向这种活性可能代表了一种阻断癌细胞中蛋白酶体功能的方法，这种方法与现有蛋白酶体抑制剂的作用机制不同。 我们建议通过高通量筛选策略和后续药物化学工作来探索这种可能性，这将使我们能够分离出阻断Rpn 11异肽酶的药物。 然后将使用培养的癌细胞测试从这一努力中产生的药物，以确定它们是否具有抗肿瘤活性。

DESCRIPTION (provided by applicant): The proteasome is the enzyme that is primarily responsible for turnover of proteins within the cytoplasm and nucleus of eukaryotic cells. A substrate destined for the proteasome is first conjugated to an ubiquitin chain, which binds to receptors on the proteasome, thereby tethering the substrate so that it can be translocated into the inner chamber of the proteasome where it is degraded. Many clinical studies have confirmed that small molecules that block the protein- degrading active sites in the inner chamber of the proteasome can stem the progression of human cancers including multiple myeloma and mantle cell lymphoma. In prior work, we discovered that the Rpn11 subunit of the proteasome contains a novel zinc metalloprotease active site that removes the ubiquitin chain from proteasome substrates. This 'deubiquitination' catalyzed by Rpn11 is essential for the protein-degrading activity of the proteasome, presumably because the bulky ubiquitin chain prevents substrate from being inserted into the inner chamber of the proteasome. Rpn11 metalloprotease thus represents a new proteasome activity that can potentially be targeted by small molecules to block degradation of proteasome substrates and stem the growth of human tumors. The goal of this application is to use this assay to screen a large compound library (~330,000 compounds) from the MLPCN located at the Conrad Prebys Center for Chemical Genomics to identify Rpn11 inhibitors and triage these inhibitors in secondary and tertiary assays devised to prioritize potent, selective, competitive, reversible and cell permeable hits. Our strategy is based on a substrate comprising four tandem ubiquitins fused to a peptide bearing a unique cysteine that can be labeled with a fluorophore. Cleavage of this substrate by Rpn11 at the junction between the fourth ubiquitin and the peptide releases the low molecular weight fluorescent peptide, resulting in a large depolarization of the fluorophore. By measuring the fluorescence polarization of the substrate in the presence of 26S proteasome plus various compounds, we should be able to identify those compounds, termed hits, that block Rpn11-dependent release of the fluorescent peptide product. Hits that subsequently pass the battery of secondary and tertiary assays will inform follow-up SAR studies to improve compound potency, selectivity and cell-permeability. These inhibitors will be employed to differentiate ubiquitin-dependent and ubiquitin-independent substrates of the proteasome and to test whether inhibition of Rpn11 is a viable strategy for chemotherapy of cancer. PUBLIC HEALTH RELEVANCE: The proteasome is the target for new drugs - 'proteasome inhibitors' - that are used to treat the hematological malignancies multiple myeloma and mantle cell lymphoma. Several years ago, we discovered a new activity of the proteasome that we named Rpn11 isopeptidase. Targeting this activity with drugs could represent a way to block proteasome function in cancer cells that differs from the mechanism of action of the existing proteasome inhibitors. We propose to explore this possibility by a high throughput screening strategy and follow-up medicinal chemistry efforts that will allow us to isolate drugs that block Rpn11 isopeptidase. Drugs that emerge from this effort will then be tested to see if they possess anti-tumor activity using cancer cells grown in culture.