High throughput screening for modulators of UBC12

描述（由申请人提供）：UBC12是人类泛素偶联酶（E2）家族的50多个成员之一，其共价将泛素样蛋白NEDD8转移到蛋白质上。它的主要底物是cullins， cullins在称为cullin-RING泛素连接酶（CRLs）的多亚基酶的背景下起作用。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。蛋白酶体降解的所有细胞蛋白中约有20%是由crl引起的，而由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及其相关的调节机制在开发人类治疗方面受到了相当大的关注。本申请的总体目标是从MLPCN中筛选一个大的化合物文库，以鉴定UBC12的调节剂，并在二级和三级分析中对这些抑制剂进行优先排序，以对初级命中的效力、选择性和细胞渗透性进行分类。我们的方法采用了我们开发并验证的TR-FRET生化试验，重建了NEDD8从UBC12转移到cullin CUL1的特定赖氨酸残基上。我们屏幕上的主要点击将被优先处理

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.