High throughput screening for modulators of UBC12

描述（由申请人提供）：UBC 12是人泛素缀合酶（E2）家族的50多个成员之一，其将泛素样蛋白NEDD 8共价转移到蛋白质上。其主要底物是cullin，cullin在称为cullin-RING泛素连接酶（CRL）的多亚基酶的背景下发挥作用。NEDD 8修饰激活CRL以刺激它们在其结合的蛋白质底物上合成泛素聚合物的能力。CRL占蛋白酶体所有细胞蛋白质降解的约20%，并且由CRL控制的调节机制的改变与诸如癌症和神经退行性疾病、炎症和免疫应答调节以及病毒和细菌感染等疾病相关。由于其在蛋白质稳态中的重要功能和对人类健康的直接影响，CRL及其相关调控机制在开发人类治疗药物方面受到了相当大的关注。本申请的总体目标是从MLPCN中筛选大的化合物文库以鉴定UBC 12的调节剂，并在二级和三级测定中优先考虑这些抑制剂，所述二级和三级测定设计用于对初级命中的效力、选择性和细胞渗透性进行分类。我们的方法采用了TR-FRET生物化学测定，我们已经开发和验证，重建NEDD 8从UBC 12转移到cullin CUL 1的特定赖氨酸残基上。从我们的屏幕上的主要命中将优先 基于已知的化学特征，以及设计用于检测UBC 12选择性、细胞渗透性和对癌细胞的效力的二级和三级测定。这些将为后续SAR提供信息，以改善体外和经处理细胞的靶向效应。从我们的研究中出现的探针有望产生新的工具，用于对UBC 12进行详细的生化和结构研究，并为cullin neddylation和CRL的调节提供前所未有的见解。我们预计这些努力将刺激E2选择性探针的发展，以阐明泛素和泛素样蛋白修饰系统的功能和调节，从而验证这些酶作为直接有益于人类健康的治疗靶点。

DESCRIPTION (provided by applicant): UBC12 is one of 50+ members of the human ubiquitin conjugating enzyme (E2) family that covalently transfers the ubiquitin-like protein NEDD8 onto proteins. Its major substrates are cullins which function in the context of multi-subunit enzymes known as cullin-RING ubiquitin ligases (CRLs). NEDD8 modification activates CRLs to stimulate their ability to synthesize ubiquitin polymers on their bound protein substrates. CRLs account for ~20% of all cellular protein degradation by the proteasome and alterations in regulatory mechanisms controlled by CRLs are associated with diseases such as cancer and neurodegerative disorders, inflammation and immune response modulation, and viral and bacterial infections. As a result of their important functions in protein homeostasis and direct impact on human health, CRLs and associated regulatory mechanisms have received considerable attention for developing human therapeutics. The overall goal of this application is to screen a large compound library from the MLPCN to identify modulators of UBC12 and prioritize these inhibitors in secondary and tertiary assays devised to categorize the potency, selectivity, and cell permeability of primary hits. Our approach employs a TR-FRET biochemical assay we have developed and validated that reconstitutes the transfer of NEDD8 from UBC12 onto a specific lysine residue of the cullin CUL1. Primary hits from our screen will be prioritized based on known chemical features and in secondary and tertiary assays designed to examine UBC12 selectivity, cell permeability, and potency on cancer cells. These will inform subsequent SAR to improve on-target effects both in vitro and on treated cells. Probes emerging from our study are expected to yield novel tools for detailed biochemical and structural studies on UBC12 as well as provide unprecedented insight into the regulation of cullin neddylation and CRLs. We anticipate these efforts will stimulate the development of E2 selective probes to elucidate the functions and regulation of ubiquitin and ubiquitin-like protein modification systems, leading to the validation of these enzymes as a therapeutic target class to directly benefit human health.