A Screen for Inhibitors of Csn-mediated Deneddylation of Cullin-Ring Ligases

描述(由申请人提供)：通过将泛素附着到细胞蛋白质上的蛋白质修饰是调节许多生物过程的关键机制。泛素通过其C末端与受体底物的赖氨酸残基之间的异肽键共价连接到靶蛋白上。另外的泛素可以连接到泛素的任何伯胺上，以在底物上形成多泛素链。通过泛素的Lys48连接在一起的=4泛素链的组装标志着细胞蛋白质被26S蛋白酶体降解。蛋白质的泛素化是通过涉及泛素激活(E1)、泛素结合(E2)和泛素连接(E3)酶的一系列酶来实现的。泛素化发生时，E3与底物结合，与泛素硫酯化的E2(E2~Ub)结合，使它们接近，从而使泛素从E2转移到底物。在研究最深入的E3中，是cullin环连接酶(CRL)超家族的成员，它们由cullin与泛素样蛋白Nedd8可逆的共价修饰来调节。Csn5是COP9-Signalosome(CSN)的一个新的金属异肽酶亚基，催化从库林斯细胞中脱除Nedd8。CRL家族有多达350个成员，其广泛的范围提出了深刻的问题，即Nedd8结合和 协调去共轭周期以实现对单个CRL的特定调节。事实上，必须有某种机制来控制Nedd8的去结合，以避免所有CRL同时失活，这可能会对细胞调节产生深远和相互冲突的影响。对Nedd8激活酶抑制剂MLN4924的研究表明，Nedd8结合-去结合的周期非常快；在给药5分钟内，几乎所有Nedd8修饰的库林斯都被去结合。因此，为了真正理解去动力的作用，在CSN被抑制后的几分钟内观察细胞将是至关重要的。要达到这个时间分辨率，遗传方法是不够的，必须有一个小分子抑制剂。本文提出的实现这一目标的具体目标是：(1)完成目前的荧光偏振96孔基线分析的发展，以使其成为功能强大、可重复性好的384孔或1536孔分析，其S/B、CV和Z‘参数的值适合支持高温超导；(2)高温超导检测方法的配置，包括：(A)建立反筛选和二次检测方法，以及(B)使用1280化合物LOPAC收集来验证一次检测。

DESCRIPTION (provided by applicant): Protein modification by the attachment of ubiquitin to cellular proteins is a key mechanism in regulating many biological processes. Ubiquitin is covalently attached to target proteins via an isopeptide bond between its C-terminus and a lysine residue of the acceptor substrate. Additional ubiquitins can be conjugated to any of the primary amines of ubiquitin to form a polyubiquitin chain on the substrate. Assembly of a chain of =4 ubiquitins linked together via Lys48 of ubiquitin marks cellular proteins for degradation by the 26S proteasome. Ubiquitination of proteins is achieved through an enzymatic cascade involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitinligating (E3) enzymes. Ubiquitination occurs when an E3 binds to both substrate and an E2 thioesterified with ubiquitin (E2~Ub), bringing them in proximity so that the ubiquitin is transferred from E2 to substrate. Among the most intensively studied E3s are members of the cullin-RING ligase (CRL) superfamily, which are regulated by a reversible covalent modification of the cullin with the ubiquitin-like protein, Nedd8. Deconjugation of Nedd8 from cullins is catalyzed by Csn5, the novel metalloisopeptidase subunit of the COP9-Signalosome (CSN). The breadth of the CRL family, which contains up to 350 members, raises profound questions about how the Nedd8 conjugation and deconjugation cycle is coordinated to effect specific regulation of individual CRLs. Indeed, there must be some mechanism to control deconjugation of Nedd8 to avoid simultaneous inactivation of all CRLs, which could be expected to have far-reaching and conflicting effects on cell regulation. Studies with the Nedd8-activating enzyme inhibitor MLN4924 reveal that the cycle of Nedd8 conjugation-deconjugation is extremely fast; within 5 minutes of administration of MLN4924, almost all of the Nedd8-modified cullins are deconjugated. Therefore, to truly understand the role of deneddylation it will be crucial to look at cells in the minutes following inhibition of CSN. To achieve this time resolution, genetic approaches are insufficient and it is essential to have a small molecule inhibitor. The specific aims proposed herein to achieve this goal are: (1) Assay development to complete transformation of the current fluorescence polarization 96-well baseline assay into a robust and reproducible 384 or 1536-well assay with values for S/B, CV, and Z' parameters suitable to support HTS and (2) Configuration of assays for HTS, including: (a) developing counter-screen and secondary assays and (b) validating the primary assay with the 1280-compound LOPAC collection.