HTS for Rpn11 chemical probes

描述(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。蛋白酶体的底物首先与泛素链连接，泛素链与蛋白酶体上的受体结合，从而将底物捆绑在一起，使其能够转移到蛋白酶体的内室，在那里被降解。许多临床研究已经证实，阻断蛋白酶体内腔蛋白降解活性部位的小分子可以阻止包括多发性骨髓瘤和套细胞淋巴瘤在内的人类癌症的进展。在以前的工作中，我们发现蛋白酶体的Rpn11亚基包含一个新的锌金属蛋白酶活性部位，它可以从蛋白酶体底物中移除泛素链。这种由Rpn11催化的‘去泛素化’对于蛋白酶体的蛋白质降解活性是必不可少的，这可能是因为巨大的泛素链阻止底物被插入蛋白酶体的内腔。因此，Rpn11金属蛋白酶代表了一种新的蛋白酶体活性，可能被小分子靶向来阻止蛋白酶体底物的降解和阻止人类肿瘤的生长。本应用程序的目标是使用此方法从位于Conrad Prebys化学基因组中心的MLPCN中筛选一个大型化合物文库(约330,000个化合物)，以识别Rpn11抑制剂，并在设计的二次和三次检测中对这些抑制剂进行分类，以优先选择有效的、选择性的、竞争性的、可逆的和细胞通透性的靶标。我们的策略是基于一种底物，该底物由四个串联泛素融合到一个含有可用荧光团标记的独特半胱氨酸的多肽上。在第四泛素和多肽的连接处被Rpn11裂解这个底物释放出低分子荧光肽，导致荧光团的大量去极化。通过测量底物在26S蛋白酶体和各种化合物存在下的荧光偏振，我们应该能够识别那些称为HITS的化合物，这些化合物可以阻止Rpn11依赖的荧光肽产物的释放。随后通过二次和三次检测的HITS将为后续的SAR研究提供信息，以提高化合物的效力、选择性和细胞渗透性。这些抑制剂将用于区分泛素依赖和泛素非依赖的蛋白酶体底物，并测试抑制Rpn11是否是癌症化疗的可行策略。 公共卫生相关性：蛋白酶体是用于治疗血液系统恶性肿瘤、多发性骨髓瘤和套细胞淋巴瘤的新药的靶点。几年前，我们发现了一种新的蛋白酶体活性，我们将其命名为Rpn11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努力来探索这种可能性，这将使我们能够分离出阻断Rpn11异肽酶的药物。通过这一努力产生的药物将被测试，看看它们是否具有抗肿瘤活性，使用在培养中生长的癌细胞。

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.