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

描述（由申请人提供）：通过将泛素连接到细胞蛋白质来进行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-末端和受体底物的赖氨酸残基之间的异肽键共价连接至靶蛋白。另外的遍在蛋白可以与遍在蛋白的任何伯胺缀合以在底物上形成聚遍在蛋白链。通过泛素的Lys 48连接在一起的=4个泛素的链的组装标志着细胞蛋白质被26 S蛋白酶体降解。蛋白质的泛素化是通过涉及泛素激活（E1）、泛素缀合（E2）和泛素连接（E3）酶的酶级联反应来实现的。 当E3与底物和与泛素硫代酯化的E2（E2~Ub）结合时，发生泛素化，使它们接近，使得泛素从E2转移到底物。其中最深入研究的E3是cullin-RING连接酶（CRL）超家族的成员，其通过用泛素样蛋白Nedd 8可逆共价修饰cullin来调节。Csn 5是C 0 P9-信号体（CSN）的新型金属异肽酶亚基，其催化Nedd 8从cullin的解缀合。CRL家族包含多达350个成员，其广度引发了关于Nedd 8共轭和 协调解偶联周期以实现对各个CRL的特定调节。事实上，必须有某种机制来控制Nedd 8的去缀合，以避免所有CRL的同时失活，这可能会对细胞调控产生深远而矛盾的影响。使用Nedd 8激活酶抑制剂MLN 4924的研究表明，Nedd 8缀合-解缀合的周期极快;在给予MLN 4924后5分钟内，几乎所有Nedd 8修饰的cullin都被解缀合。因此，为了真正理解去eddylation的作用，在CSN抑制后的几分钟内观察细胞将是至关重要的。为了实现这种时间分辨率，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的是：（1）试验开发，以完成当前荧光偏振96孔基线试验向稳健且可再现的384或1536孔试验的转化，其中S/B、CV和Z'参数的值适合于支持HTS，以及（2）HTS试验的配置，包括：（a）开发反筛选和二次测定和（B）用1280-化合物LOPAC收集物验证初级测定。

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.