Novel Screening Platform for Discovery of DUB Targeting Probes

摘要 去泛素化酶（DUBs）家族在几乎所有的生理和病理过程中发挥关键作用。 生理过程。重要的是DUB功能的破坏与广泛的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 DUB的抑制剂，没有专门的化合物库集中在这些重要的酶。 我们跨学科团队的目标（Sara Buhrlage，DUB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，验证我们的新的， DUB靶向化合物的cused库。在充分的时间，从我们的建议结果将推动发展- 新型有效和选择性共价DUB抑制剂的开发以及DUB在细胞和动物中的功能研究 模型 与PAR-17 - 438一致，我们专注于检测试剂盒开发，重点是结合多种或- 正交生化测定作为关键验证步骤。我们将通过三个方面开发和验证我们的平台 具体目标： 目标1.开发和执行一个由49个DUB和一个重点收集器组成的图书馆与图书馆屏幕， 150种DUB靶向化合物的反应。我们将使用基于泛素的DUB活性探针（ABPs）进行竞争， 使用来自我们的DUB靶向化合物库的化合物进行的定量形式测定。MS定量分析 这些测定将建立我们的化合物对细胞DUB的选择性分布。我们将使用基于细胞的 和完整蛋白CE-MS以确认中靶活性和共价结合。 目标二。绘制不可逆DUB靶向化合物的蛋白质组范围反应性。我们将使用两个 新的基于质谱的MS方法，以进一步确认共价靶-配体结合，以及 确定每个靶标上的特定修饰位点。这些分析将提供浓度的直接读数， DUB上的离子依赖性探针结合以及整个蛋白质组中潜在的脱靶半胱氨酸。作为 因此，这些分析也将作为我们化合物的全面反筛选。 目标3：验证我们平台简化药物化学优化活动的能力。在 为此，我们将验证我们的平台量化晚期DUB探针选择性变化的能力 通过结构-活性关系（SAR）研究进行优化。 数据和结果，从我们的建议将提供（i）一个库的良好表征的共价DUB化合物;（ii） 一个广泛可用的资源，包括公开可用的协议和准则;（三）一个共同的生物分析 适用于其他酶家族的框架;以及（iv）用于将来开发成选择性探针的命中物， 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.