Chemical Biology of the Control of Neddylation by DCN1

摘要 长期目标是产生和使用互补的化学和生物探针来研究挑选 环泛素连接酶（CRL的），并了解其激活控制的相互作用的缺陷 在Cullin Neddylation 1（DCN 1）和UBE 2 M蛋白中。因为CRL最终控制了许多 不同的蛋白质，从而调节其稳定性，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调节关键细胞凋亡的机制。 信号网络和驱动疾病进展。该项目的健康相关性在于两个事实：1） DCN 1是一种癌蛋白，在鳞状细胞癌中扩增，驱动高度恶性表型， 2)CRL驱动的泛素化是多种疾病的有效靶点，特别是癌症和免疫性疾病。 功能障碍因此，有效的、选择性的和生物可利用的DCN 1-UB 2 M相互作用的抑制剂具有 作为抗肿瘤药物和可能用于其他疾病的潜力。泛素化受以下因素调节： 高度复杂、动态和冗余的网络。DCN 1-UB 2 M的抑制剂将允许直接询问 网络子部分的功能，并可能揭示该法规的基本原则 泛素化互补的细胞和小鼠遗传模型的产生将使独立的 验证假设。最后，DCN 1-UBE 2 M相互作用需要UBE 2 M的N-末端乙酰化， 控制蛋白质相互作用的常见翻译后修饰。因此，针对N- 末端乙酰化依赖的蛋白质相互作用可以广泛应用。研究设计和 实现这些目标的方法包括结构驱动的、假设的、 基础药物化学;亲和力和抑制效力的体外生物化学测量; 化合物功效和药效学反应;以及化合物的体外和体内测量 生物利用度、分布、代谢、排泄和毒性。总体目标是开发新的互补性 化学和生物学工具来了解泛素样蛋白NEDD 8的调节，并揭示 DCN介导neddylation在Cullin-RING连接酶底物受体交换、生长因子 信号传导和驱动肿瘤进展。我们的目标是：目标1：提高我们的效力和口服暴露 目前的DCN 1/2抑制剂，并产生新的化学探针，具有足够的效力和选择性， 我们自己和其他人研究抑制细胞和动物中E2-E3相互作用的后果。目标2. 研究细胞CUL 1和CUL 3连接酶的组成如何动态响应环境 DCN 1/2在这一过程中的重要性。目的3：使用遗传和药理学 在动物肿瘤模型中研究抑制DCN 1活性的效果的方法。

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.