Chemical Biology of the Control of Neddylation by DCN1

摘要 长期目标是产生和使用免费的化学和生物探针来研究扑杀 环泛素连接酶(CRL)并了解其激活受缺陷者相互作用的控制 在Cullin Neddyling1(DCN1)和UBE2M蛋白中。因为CRL最终控制着许多 不同的蛋白质，从而调节它们的稳定性、细胞内定位和功能，具有时空 对DCN介导的CRL活性的控制有可能解开关键细胞的调节机制 信号网络和推动疾病进展。这个项目与健康的相关性在于两个事实：1) DCN1是一种癌蛋白，在鳞癌中扩增，导致高度恶性的表型，并且 2)CRL驱动的泛素化是多种疾病，特别是癌症和免疫疾病的有效靶点 功能障碍。因此，有效的、选择性的和生物可利用的DCN1-UB2M相互作用的抑制剂 有潜力被开发为抗肿瘤药物，并可能用于其他疾病。泛素化受以下因素调节 高度复杂、动态且冗余的网络。DCN1-UB2M的抑制剂将允许直接询问 网络的子部分的功能，并可能揭示监管的基本原则 泛素化。互补的细胞和小鼠遗传模型的产生将使独立 对假设的验证。最后，DCN1-UBE2M的相互作用需要UBE2M的N-末端乙酰化，a 控制蛋白质相互作用的常见翻译后修饰。因此，针对N-的战略-- 依赖末端乙酰化的蛋白质相互作用具有广泛的应用前景。本研究的设计与实现 实现这些目标的方法包括综合和递归地使用结构驱动、假设和 基础药物化学.亲和力和抑制力的体外生物化学测量.体内测量 复方药效、药效学及体内外测定 生物利用度、分布、代谢、排泄和毒性。总体目标是开发新的免费赠品 化学和生物工具，以了解泛素样蛋白NEDD8的调节，并揭示 DCN介导的转录在连接酶底物受体交换、生长因子中的特异性作用 信号，并推动肿瘤的进展。我们的目标是：目标1：提高我们的 目前的DCN1/2抑制剂，并产生具有足够效力和选择性的新化学探针，以使 我们和其他人研究在细胞和动物中抑制这种E2-E3相互作用的后果。目标2. 研究细胞内CUL1和CUL3连接酶的组成对环境的动态响应 以及DCN1/2在这一过程中的重要性。目标3：使用遗传学和药理学 在动物肿瘤模型中研究抑制DCN1活性的方法。

Abstract The long-term goal is to generate and use complimentary chemical and biological probes to study the cullin RING ubiquitin ligases (CRL’s) and understand their activation controlled by the interaction of by the Defective in Cullin Neddylation 1 (DCN1) and UBE2M proteins. Because the CRL’s ultimately control ubiquitination of many diverse proteins, thus regulating their stability, intracellular localization, and function, having spatiotemporal control over DCN-mediated CRL activity has the potential to unravel the mechanism regulating key cellular signaling networks and driving disease progression. The health relatedness of this project lies in two facts: 1) DCN1 is an oncoprotein, amplified in squamous cell carcinomas, that drives a highly malignant phenotype, and 2) CRL driven ubiquitination is a validated target in multiple diseases, particularly cancer and immune dysfunction. Therefore, inhibitors of the DCN1-UB2M interaction that are potent, selective, and bioavailable have the potential to be developed as antitumor drugs and possibly for other diseases. Ubiquitination is regulated by a highly complex, dynamic, and redundant network. Inhibitors of DCN1-UB2M will allow direct interrogation of the function of sub-portions of the network and are likely to unveil fundamental principles of the regulation ubiquitination. The generation of complementary cellular and mouse genetic models will enable independent verification of hypotheses. Finally, the DCN1-UBE2M interaction requires N-terminal acetylation of UBE2M, a common posttranslational modification controlling protein interactions. Therefore, a strategy for targeting N- terminal acetylation dependent protein interactions could be widely applicable. The research design and methods for achieving these goals involves the integrated and recursive use of structure-driven, hypothesis- based medicinal chemistry; in vitro biochemical measures of affinity and inhibitory potency; in vivo measures of compound efficacy and pharmacodynamic responses; and in vitro and in vivo measures of compound bioavailability, distribution, metabolism, excretion, and toxicity. The overall goal is to develop new complimentary chemical and biological tools to understand the regulation of the ubiquitin-like protein NEDD8, and uncover the specific role of DCN-mediated neddylation in Cullin-RING ligase substrate receptor exchange, growth factor signaling, and driving tumor progression. Our aims are: Aim 1: Improve the potency and oral exposure of our current DCN1/2 inhibitors and generate new chemical probes with sufficient potency and selectivity to enable ourselves and others to study the consequences of inhibiting this E2-E3 interaction in cells and animals. Aim 2. Investigate how the composition of cellular CUL1 and CUL3 ligases dynamically responds to environmental perturbations and the importance of DCN1/2 in this process. Aim 3: Use genetic and pharmacological approaches to study the effects of inhibiting DCN1 activity in animal tumor models.