High throughput screening for modulators of UBC12

UBC12 调节剂的高通量筛选

基本信息

批准号：
8328053
负责人：
Matthew Petroski
金额：
$ 4.88万
依托单位：
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8328053
关键词：
Accounting Advanced Development Attention Bacterial Infections Binding Binding Proteins Binding Sites Biochemical Biological Assay CUL1 gene Cancer cell line Cell Death Cell Death Induction Cell Survival Cells Chemical Structure Chemicals Colon Carcinoma Cullin Proteins DNA Development Disease Dose Enzymes Family Fluorescence Resonance Energy Transfer Future Goals HCT116 Cells Health Homeostasis Human Human Ubiquitin Immune response In Vitro Inflammation Inhibitory Concentration 50 Libraries Link Lysine Malignant Epithelial Cell Malignant Neoplasms Measures Modification Neurodegenerative Disorders Pathway interactions Permeability Polymers Post-Translational Protein Processing Proteins Regulation Role S Phase Screening procedure Site Structure Sulfhydryl Compounds System Testing Therapeutic Translations Ubiquitin Ubiquitin Like Proteins Ubiquitin-Conjugating Enzymes Validation Virus Diseases Work Xenograft procedure base cancer cell design high throughput screening human disease improved inhibitor/antagonist insight member multicatalytic endopeptidase complex novel novel strategies pathogen prevent protein degradation reconstitution research study response small molecule therapeutic target tool tumor growth ubiquitin ligase

项目摘要

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.

描述（由申请人提供）：UBC12 是人类泛素结合酶 (E2) 家族 50 多个成员之一，该家族将泛素样蛋白 NEDD8 共价转移到蛋白质上。其主要底物是 cullin，它在称为 cullin-RING 泛素连接酶 (CRL) 的多亚基酶的背景下发挥作用。 NEDD8 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 约占蛋白酶体所有细胞蛋白降解的 20%，并且 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相关。由于它们在蛋白质稳态中的重要功能以及对人类健康的直接影响，CRL 和相关的调节机制在开发人类疗法方面受到了相当多的关注。该应用的总体目标是从 MLPCN 中筛选大型化合物库，以识别 UBC12 调节剂，并在二级和三级测定中对这些抑制剂进行优先级排序，这些测定旨在对主要命中的效力、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 生化测定，该测定可重构 NEDD8 从 UBC12 到 cullin CUL1 的特定赖氨酸残基上的转移。我们屏幕上的主要点击将被优先考虑基于已知的化学特征以及旨在检查 UBC12 选择性、细胞渗透性和对癌细胞的效力的二级和三级测定。这些将为后续的 SAR 提供信息，以改善体外和处理细胞上的靶向效果。我们的研究中出现的探针预计将为 UBC12 的详细生化和结构研究提供新的工具，并为 cullin neddylation 和 CRL 的调控提供前所未有的见解。我们预计这些努力将刺激 E2 选择性探针的开发，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治疗靶标类别的有效性，从而直接造福人类健康。