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细胞质和细胞核内蛋白质周转的酶。到达蛋白酶体的底物首先与泛素链结合，泛素链与蛋白酶体上的受体结合，从而将底物捆绑在一起，使底物能够被转运到蛋白酶体的内腔中，在那里被降解。许多临床研究已经证实，阻断蛋白酶体内室中蛋白质降解活性位点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。在之前的工作中，我们发现蛋白酶体的Rpn11亚基含有一个新的锌金属蛋白酶活性位点，可以从蛋白酶体底物中去除泛素链。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，可能是因为庞大的泛素链阻止底物插入蛋白酶体的内室。因此，Rpn11金属蛋白酶代表了一种新的蛋白酶体活性，可以被小分子靶向以阻止蛋白酶体底物的降解和阻止人类肿瘤的生长。该应用程序的目的是使用该方法筛选位于Conrad Prebys化学基因组学中心的MLPCN中的大型化合物库（约33万个化合物），以识别Rpn11抑制剂，并在二级和三级分析中对这些抑制剂进行分类，以优先考虑有效的、选择性的、竞争性的、可逆的和细胞渗透性的命中。我们的策略是基于一种底物，该底物由四种串联泛素融合到一种具有独特半胱氨酸的肽上，这种肽可以用荧光团标记。Rpn11在第四泛素和肽之间的交界处切割该底物，释放低分子量荧光肽，导致荧光团大量去极化。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被称为hit的化合物，这些化合物阻断了依赖于rpn11的荧光肽产物的释放。随后通过一系列二级和三级分析的命中将为后续的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蛋白酶体的泛素依赖性底物和非泛素依赖性底物，并测试抑制Rpn11是否是癌症化疗的可行策略。

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.