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: 10396625
• 负责人: Sara J Buhrlage
• 金额: $ 59.16万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396625
• 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; 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）的家族在几乎所有生理和病情中执行关键功能 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，感染和神经退行性。但是，目前很少有选择性 配音的抑制剂和没有专门的化合物文库的抑制剂集中在这些重要的酶上。 我们的跨学科团队的目标（Sara Buhrlage，Dub Biology，Chemical Biology; Jarrod Marto蛋白Bi- 化学蛋白质组学的ochemistrion）是开发一个可靠的筛选平台，对我们的小说fo-fo-进行了验证 将DUB靶向化合物的库。在大量的时间里，我们的提案将推动发展 - 新的有效和选择性的共价配音抑制剂以及在细胞和动物中的配音功能的研究 型号。 与Par-17-438一致，我们专注于测定开发，重点是纳入多个，或 - Thogonal生化测定作为关键验证步骤。我们将通过三个 具体目的： AIM 1。开发和执行由49个配音组成的图书馆-VS图书馆图书馆，并集中的collec- 150种靶向化合物的含量。我们将在竞争中使用基于泛素的DUB活动探针（ABP） 带有来自DUB靶向化合物库的化合物的TION格式测定法。定量MS分析 这些测定将建立我们化合物针对细胞配音的选择性曲线。我们将使用基于细胞的 和完整的蛋白质CE-MS确认靶向活性和共价结合。 目标2。绘制不可逆的靶向化合​​物的全蛋白质组反应性。我们将使用两个 基于新型质谱的新型MS方法，以进一步确认共价靶 - 配体结合，以及 确定每个目标上修改的特定位点。这些测定将提供浓度的直接读数 在整个蛋白质组中，依赖于配音以及潜在的脱靶半胱氨酸的探针结合。作为 结果，这些测定也将作为我们化合物的全面柜台屏幕。 目标3。验证我们平台简化药物化学优化活动的能力。在 这个目标我们将验证平台量化后期配音选择性变化的能力 通过结构活性关系（SAR）研究进行了完善。 我们的提案的数据和结果将提供（i）特征良好的共价配音库； （ii） 具有公共协议和准则的广泛访问资源； （iii）常见的生物分析 适用于其他酶家庭的框架； （iv）将未来开发的命中作为选择性探针和 NCI实验疗法（下一个）计划的潜在候选者。