High throughput screening for modulators of UBC12

描述(申请人提供)：UBC12是人类泛素结合酶(E2)家族的50多个成员之一，它能将泛素样蛋白NEDD8共价转移到蛋白质上。它的主要底物是库林酶，它在多亚单位酶的背景下发挥作用，称为卡尔林环泛素连接酶(CRL)。NEDD8修饰激活CRL，刺激它们在结合蛋白底物上合成泛素聚合物的能力。CRL约占蛋白酶体所有细胞蛋白质降解的20%，由CRL控制的调节机制的改变与癌症和神经退行性疾病、炎症和免疫反应调节以及病毒和细菌感染等疾病有关。由于CRLS在蛋白质动态平衡和直接影响人类健康方面的重要作用，CRLS及其相关的调控机制在开发人类治疗药物方面受到了极大的关注。这项应用的总体目标是从MLPCN中筛选一个大型化合物文库，以确定UBC12的调节器，并在设计的二次和三次检测中优先考虑这些抑制剂，以分类一次击中的效力、选择性和细胞渗透性。我们的方法使用了我们开发并验证的TRRET生化分析，该方法将NEDD8从UBC12重组到cullin CUL1的特定赖氨酸残基上。我们屏幕上的主要点击将按优先顺序排列 根据已知的化学特征以及旨在检测UBC12选择性、细胞渗透性和对癌细胞效力的二次和三次检测。这些将为后续的SAR提供信息，以改善体外和处理细胞的靶向效应。我们研究中出现的探针有望为UBC12的详细生化和结构研究提供新的工具，并提供前所未有的对cullin核苷酸和CRLS的调控的洞察力。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，从而使这些酶被确认为直接有益于人类健康的治疗靶类。

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.