Novel Screening Platform for Discovery of DUB Targeting Probes

摘要 脱泛素酶家族(DUBS)在几乎所有的生理和病理过程中发挥着关键作用。 生理过程。重要的是，DUB功能的中断与广泛的HU- 人类疾病，包括癌症、感染和神经变性。然而，目前很少有选择性的 DUBS的抑制剂，没有专门的化合物文库专注于这些重要的酶。 我们跨学科团队的目标(Sara Buhrlage，配音生物学，化学生物学；Jarrod Marto蛋白质双- 化学，化学蛋白质组学)是开发一个强大的筛选平台，与我们的新Fo-Fo- 以配对为靶向的化合物文库。随着时间的推移，我们提案的结果将推动发展- 新型高效选择性共价DUB抑制剂的研制及DUB在细胞和动物中的作用研究 模特们。 与PAR-17-438一致，我们专注于分析开发，重点是结合多个或- 正交生化分析是关键的验证步骤。我们将通过三个阶段来开发和验证我们的平台 具体目标： 目标1.开发和执行由49个配音和一个聚焦的同事组成的图书馆对图书馆屏幕-- 150种双靶向化合物的合成。我们将在竞争中使用基于泛素的配对活性探针(ABPs)。 TION格式与我们的DUB靶向化合物库中的化合物进行分析。大豆油的定量MS分析 这些分析将建立我们的化合物对细胞复制的选择性分布。我们将使用基于细胞的 和完整的蛋白质CE-MS以确认靶标活性和共价结合。 目的2.绘制不可逆DUB靶向化合物的全蛋白质组反应性图谱。我们将使用两个 基于质谱学的新方法以进一步确认共价靶-配体结合，以及 确定每个目标上的特定修改位置。这些分析将提供浓度的直接读数- 依赖于TION的探针结合在DUBS上以及整个蛋白质组中潜在的脱靶半胱氨酸上。AS 因此，这些分析也将作为我们化合物的全面柜台筛选。 目的3.验证我们平台简化药物化学优化活动的能力。在……里面 为了达到这个目的，我们将验证我们的平台量化后期DUB探针选择性变化的能力 通过结构-活性关系(SAR)研究得到提纯。 我们建议的数据和结果将提供(I)一个表征良好的共价配位化合物的资料库；(Ii) 具有公开可用方案和准则的可广泛获取的资源；(3)共同的生物分析 适用于其他酶家族的框架；以及(Iv)未来开发选择性探针和 NCI实验治疗(NEXT)计划的潜在候选人。

ABSTRACT The family of deubiquitinating enzymes (DUBs) perform key functions in virtually all physiological and patho- physiological processes. Importantly disruption of DUB function has been associated with a wide range of hu- man diseases, including cancer, infection, and neurodegeneration. However, there are currently few selective inhibitors of DUBs and no dedicated compound libraries focused on these important enzymes. The goal of our interdisciplinary team (Sara Buhrlage, DUB biology, chemical biology; Jarrod Marto protein bi- ochemistry, chemical proteomics) is to develop a robust screening platform, validated against our novel, fo- cused library of DUB-targeting compounds. In the fullness of time results from our proposal will drive develop- ment of new potent and selective covalent DUB inhibitors and study of DUB function in cellular and animal models. Consistent with PAR-17-438, we focus on assay development, with emphasis on incorporating multiple, or- thogonal biochemical assays as key validation steps. We will develop and validate our platform through three specific aims: Aim 1. To develop and execute a library-vs-library screen comprised of 49 DUBs and a focused collec- tion of 150 DUB-targeting compounds. We will use ubiquitin-based DUB activity probes (ABPs) in competi- tion format assays with compounds from our library of DUB-targeting compounds. Quantitative MS analysis of these assays will establish selectivity profiles of our compounds against cellular DUBs. We will use cell-based and intact protein CE-MS to confirm on-target activity and covalent binding. Aim 2. To map the proteome-wide reactivity of irreversible DUB-targeting compounds. We will use two novel mass spectrometry-based MS approaches to further confirm covalent target-ligand binding, as well as to identify the specific site of modification on each target. These assays will provide a direct readout of concentra- tion-dependent probe binding on DUBs as well as on potential off-target cysteines throughout the proteome. As a result, these assays will also serve as comprehensive counter screens for our compounds. Aim 3. To validate our platform's ability to streamline medicinal chemistry optimization campaigns. In this Aim we will validate the ability of our platform to quantify changes in selectivity for late-stage DUB probes refined through structure-activity relationship (SAR) studies. Data and results from our proposal will provide (i) a library of well-characterized covalent DUB compounds; (ii) a broadly-accessible resource with publicly available protocols and guidelines; (iii) a common bioanalytical framework applicable to other enzyme families; and (iv) hits for future development into selective probes and potential candidates for the NCI Experimental Therapeutics (NExT) Program.