Novel Screening Platform for Discovery of DUB Targeting Probes

用于发现 DUB 靶向探针的新型筛选平台

• 批准号: 10166601
• 负责人: Sara J Buhrlage
• 金额: $ 61.09万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166601
• 关键词: Affinity; Animal Model; Animals; Binding; Biochemical; Biological Assay; Biology; Capillary Electrophoresis; Cell model; Cell physiology; Cells; Chemicals; Collection; Critical Pathways; Cysteine; Data; Deubiquitinating Enzyme; Development; Disease; Dose; Drug Targeting; Ensure; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzymes; Exhibits; Family; Family member; Feeds; Future; Genetic Screening; Goals; Guidelines; Homeostasis; Immunity; Individual; Infection; Investigational Therapies; Investments; Ions; Lead; Libraries; Ligand Binding; Ligands; MJD1 protein; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Modification; National Institute of Allergy and Infectious Disease; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Neurodegenerative Disorders; Pharmaceutical Chemistry; Physiological; Physiological Processes; Physiology; Play; Process; Protein Kinase; Proteins; Proteome; Proteomics; Protocols documentation; Reagent; Regulation; Reproducibility; Resources; Role; Scanning; Serine Hydrolase; Site; Structure; Structure-Activity Relationship; Tars; Technology; Therapeutic; Time; UCHL1 gene; Ubiquitin; Validation; Western Blotting; activity-based protein profiling; assay development; base; chemoproteomics; design; enzyme activity; human disease; improved; inhibitor/antagonist; man; member; next generation; novel; pathogen; programs; protease Re; response; scaffold; screening; small molecule; small molecule libraries; structural biology; virtual

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.

抽象的 去泛素化酶 (DUB) 家族在几乎所有的生理和病理过程中发挥着关键功能。 生理过程。重要的是，DUB 功能的破坏与多种 hu- 人类疾病，包括癌症、感染和神经退行性疾病。但目前选择性极少 DUB 抑制剂，并且没有专门针对这些重要酶的化合物库。 我们的跨学科团队（Sara Buhrlage，DUB 生物学，化学生物学；Jarrod Marto 蛋白质双- o化学，化学蛋白质组学）的目的是开发一个强大的筛选平台，针对我们的新颖的、fo-进行验证 所使用的 DUB 靶向化合物库。当时间成熟时，我们提案的结果将推动发展- 新型强效选择性共价 DUB 抑制剂的开发以及细胞和动物中 DUB 功能的研究 模型。 与 PAR-17-438 一致，我们专注于检测开发，重点是整合多种或- 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. 开发并执行由 49 个 DUB 和一个重点收集组成的库与库筛选 150 种 DUB 靶向化合物的合成。我们将在竞争中使用基于泛素的 DUB 活性探针 (ABP) 使用我们的 DUB 靶向化合物库中的化合物进行化验。定量 MS 分析 这些测定将建立我们的化合物针对细胞 DUB 的选择性特征。我们将使用基于细胞的 和完整蛋白质 CE-MS 以确认目标活性和共价结合。 目标 2. 绘制不可逆 DUB 靶向化合物的蛋白质组反应性图谱。我们将使用两个 新颖的基于质谱的 MS 方法可进一步确认共价靶标-配体结合，以及 确定每个目标上的具体修饰位点。这些测定将提供浓度的直接读数 依赖于 DUB 的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，这些测定也将作为我们化合物的综合计数器筛选。 目标 3. 验证我们的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后期 DUB 探针选择性变化的能力 通过结构-活性关系（SAR）研究进行完善。 我们提案的数据和结果将提供（i）一个特征良好的共价 DUB 化合物库； (二) 具有公开可用的协议和指南的可广泛访问的资源； (iii) 通用生物分析 适用于其他酶家族的框架； (iv) 未来发展为选择性探针的热门项目 NCI 实验治疗 (NExT) 计划的潜在候选人。