Assay Development for the Identification of NEDD8- activating Enzyme Inhibitors

鉴定 NEDD8 激活酶抑制剂的检测方法开发

• 批准号: 8977494
• 负责人: Matthew Petroski
• 金额: $ 44.61万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-03 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977494
• 关键词: Affinity; Basic Science; Binding; Biochemical; Biological; Biological Assay; Cancer Patient; Cell physiology; Cells; Chemicals; Chronic; Clinical; Clinical Data; Clinical Trials; Cullin Proteins; Data; Development; Disease; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Equilibrium; Etiology; Experimental Neoplasms; Health; Human; Inflammation; Libraries; Ligands; Lysine; Malignant Neoplasms; Measures; Mediating; Mutation; Normal Cell; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Property; Proteins; Regulation; Research; Resistance; Role; Signal Pathway; Specificity; System; Testing; Therapeutic; Therapeutic Agents; Translational Research; Ubiquitin; Ubiquitin Like Proteins; Work; adenylate; antitumor effect; assay development; base; cancer cell; cancer therapy; enzyme activity; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; insight; interest; mimetics; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; pre-clinical; preclinical study; response; screening; small molecule; small molecule inhibitor; small molecule libraries; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): The ubiquitin-like protein NEDD8 functions to regulate the activation of cullin-RING ubiquitin ligases (CRLs). Although CRLs have well-established roles in many fundamental aspects of cellular physiology, recent pre- clinical studies have found that cancer cells are more reliant on NEDD8 for proliferation and survival than normal cells. These observations have invigorated interest in devising strategies to pharmacologically inhibit NEDD8 to treat cancer and other diseases. The NEDD8-activating enzyme (NAE) represents the most attractive target of the NEDD8 system as it possess a catalytic pocket suitable for binding small molecules and undergoes structural rearrangements important for its activities. MLN4924, a mechanism-based inhibitor that generates a NEDD8-adenylate mimetic in the NAE catalytic pocket, represents the only NEDD8 system inhibitor and is currently being evaluated on cancer patients. We and other have found that cancer cells and experimental tumors rapidly lose sensitivity to MLN4924 through treatment-emergent mutations in NAE that impact inhibitor binding by altering the enzyme's biochemical properties. Although the implications of these on MLN4924 therapy are currently unknown, they suggest cancer cells tolerate considerable variability in NAE function. These mutations render cells broadly resistant to molecules targeting the enzyme's catalytic pocket. This project, submitted in response to PAR-13-364 Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery, seeks to develop highly innovative biophysical and cell-based assays focused on the NEDD8 system and to use these in pilot screens to identify proof-of-concept probes. Through our preliminary studies, we have devised a homogeneous biophysical assay that measures ligand- dependent changes in NAE thermal stability. We hypothesize that this assay will allow for different classes of equilibrium-binding reversible inhibitors to be identifie by screening chemically diverse small molecule libraries. To test this hypothesis and achieve the overall objective of the research, three Specific Aims are proposed: 1) to establish and validate biophysical assays that measure NAE thermal stability and supporting specificity assays; 2) to develop cell-based assays that measure biological effects of NAE inhibition; and 3) to conduct focused high-throughput screens with selected libraries, verify hits, and determine hit potency and selectivity. IMPACT: This work is highly innovative with translational relevance based on the extraordinary opportunity it provides to develop new research tools and therapeutic approaches to inhibit NAE and the NEDD8 system for the treatment of cancers and other diseases. Through our efforts focused on generating novel assays and chemical probes, these studies are expected to provide important new insight into the NEDD8 system and other ubiquitin and ubiquitin-like protein modification systems to improve human health.

描述（由申请人提供）：泛素样蛋白 NEDD8 的功能是调节 cullin-RING 泛素连接酶（CRL）的激活。尽管 CRL 在细胞生理学的许多基本方面具有明确的作用，但最近的临床前研究发现，癌细胞比正常细胞更依赖 NEDD8 进行增殖和生存。这些观察结果激发了人们对设计药理学抑制 NEDD8 以治疗癌症和其他疾病的策略的兴趣。 NEDD8 激活酶 (NAE) 是 NEDD8 系统中最具吸引力的靶标，因为它拥有适合结合小分子的催化口袋，并经历对其活性至关重要的结构重排。 MLN4924 是一种基于机制的抑制剂，可在 NAE 催化口袋中生成 NEDD8 腺苷酸模拟物，是唯一的 NEDD8 系统抑制剂，目前正在癌症患者身上进行评估。我们和其他人发现，癌细胞和实验性肿瘤通过 NAE 中的治疗突变迅速失去对 MLN4924 的敏感性，这些突变通过改变酶的生化特性来影响抑制剂的结合。尽管这些对 MLN4924 治疗的影响目前尚不清楚，但它们表明癌细胞可以耐受 NAE 功能的相当大的变异性。这些突变使细胞对针对酶催化袋的分子具有广泛的抵抗力。该项目是为了响应 PAR-13-364 用于探针和治疗前发现的高通量筛选测定的开发而提交的，旨在开发专注于 NEDD8 系统的高度创新的生物物理和基于细胞的测定，并使用这些测定在试点屏幕中识别概念验证探针。通过我们的初步研究，我们设计了一种均质生物物理测定法，用于测量 NAE 热稳定性的配体依赖性变化。我们假设该测定将允许通过筛选化学上不同的小分子库来鉴定不同类别的平衡结合可逆抑制剂。为了检验这一假设并实现研究的总体目标，提出了三个具体目标：1）建立并验证测量 NAE 热稳定性和支持特异性测定的生物物理测定； 2) 开发基于细胞的测定法来测量 NAE 抑制的生物效应； 3) 对选定的文库进行集中的高通量筛选，验证命中并确定命中效力和选择性。影响：这项工作具有高度创新性和转化相关性，因为它为开发新的研究工具和治疗方法来抑制 NAE 和 NEDD8 系统来治疗癌症和其他疾病提供了非凡的机会。通过我们专注于产生新的检测方法和化学探针的努力，这些研究预计将为 NEDD8 系统和其他泛素和泛素样蛋白质修饰系统提供重要的新见解，以改善人类健康。