Assay Development for the Identification of NEDD8- activating Enzyme Inhibitors

描述(申请人提供)：泛素样蛋白NEDD8的功能是调节剔除环泛素连接酶(CRL)的激活。尽管CRL在细胞生理学的许多基本方面都有公认的作用，但最近的临床前研究发现，癌细胞比正常细胞更依赖NEDD8进行增殖和存活。这些观察结果激发了人们对设计药物抑制NEDD8治疗癌症和其他疾病的策略的兴趣。NEDD8激活酶(NAE)是NEDD8系统中最有吸引力的靶标，因为它具有适合结合小分子的催化口袋，并经历了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，在NAE催化口袋中产生NEDD8-腺苷模拟物，是唯一的NEDD8系统抑制剂，目前正在癌症患者身上进行评估。我们和其他人已经发现，癌细胞和实验性肿瘤通过治疗出现的NAE突变，通过改变酶的生化特性来影响抑制剂的结合，从而迅速失去对MLN4924的敏感性。尽管这些对MLN4924治疗的影响目前尚不清楚，但它们表明癌细胞耐受NAE功能的相当大的变异性。这些突变使细胞对靶向酶催化口袋的分子产生广泛的抵抗力。该项目是应PAR-13-364《高通量筛选分析的开发》提交的，用于探针和治疗前发现，旨在开发以NEDD8系统为重点的高度创新的生物物理和基于细胞的分析，并在试点筛选中使用这些方法来识别概念验证探针。通过我们的初步研究，我们设计了一种均相生物物理方法，测量NAE热稳定性随配体的变化。我们假设这项试验将允许通过筛选化学上不同的小分子文库来鉴定不同类别的平衡结合的可逆抑制物。为了验证这一假设并实现研究的总体目标，提出了三个具体目标：1)建立和验证衡量NAE热稳定性的生物物理分析方法，并支持特异性分析；2)开发基于细胞的分析方法，测量NAE抑制的生物学效应；以及3)利用选定的文库进行有针对性的高通量筛选，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，具有翻译相关性，因为它提供了开发新的研究工具和治疗方法的特殊机会，以抑制NAE和NEDD8系统，用于治疗癌症和其他疾病。通过我们专注于开发新的分析和化学探针的努力，这些研究有望为NEDD8系统和其他泛素和泛素样蛋白修饰系统提供重要的新见解，以改善人类健康。

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.