Tools for DUB Drug Discovery

DUB 药物发现工具

• 批准号: 10159220
• 负责人: Sara J Buhrlage
• 金额: $ 32.39万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159220
• 关键词: Address; Antineoplastic Agents; Architecture; Behavior; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biology; Bortezomib; Cell membrane; Cell physiology; Cells; Chemicals; Clinical; Collaborations; Collection; Competitive Binding; Complement; Data; Data Analyses; Deubiquitinating Enzyme; Disease; Drug Targeting; Enzyme Inhibitor Drugs; Enzymes; FDA approved; Family; Foundations; Goals; Hematologic Neoplasms; Human; Joints; Lead; Libraries; Link; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Multiple Myeloma; Oncogenic; Oncoproteins; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Physiological; Proteasome Inhibitor; Protein Family; Proteins; Proteome; Proteomics; Reagent; Regulation; Reproducibility; Role; Serine Hydrolase; Structure; System; Technology; Testing; Therapeutic Effect; Tissues; Toxic effect; Ubiquitin; Velcade; Vent; Work; activity-based protein profiling; analytical method; analytical tool; base; blind; bropirimine; cancer cell; cancer therapy; chemoproteomics; design; drug discovery; enzyme activity; improved; inhibitor/antagonist; instrument; interest; member; multicatalytic endopeptidase complex; new technology; new therapeutic target; novel; protein degradation; small molecule; small molecule inhibitor; small molecule libraries; success; therapeutic target; tool

ABSTRACT The ubiquitin-proteasome system (UPS) has emerged over the past 15 years as an exciting new druggable pathway for cancer therapy. The family of 100 deubiquitinating enzymes (DUBs) remove ubiquitin from sub- strate proteins. Recent studies suggest that pharmacologic inhibition of DUBs may provide an opportunity to therapeutically target oncoproteins in cancer with improved precision. Despite increasing interest in their func- tion and potential as therapeutic targets, there are limited reagents and analytical platforms to study DUBs. The goal of our interdisciplinary team (Sara Buhrlage, DUB biology, chemical biology; Jarrod Marto protein bi- ochemistry, chemical proteomics) is to develop novel DUB-focused activity reagents and related analytical methods. Achieving our milestones will directly support near-term (3-5 years) efforts by us and others in the field to develop selective tool compounds to interrogate basic DUB biology and further validate specific DUBs as cancer drug targets. We will pursue our objectives through two specific aims: Aim 1. To develop and characterize novel DUB activity-based probes (ABPs) for use in activity-based protein profiling (DUB-ABPP). We will develop an expanded collection of DUB-ABPs, including PTM- modified ubiquitin. We will validate each ABP to confirm their ability to selectively engage and enrich a subset of cellular DUBs. Probes which meet this milestone will be used in combination in multiplexed, mass spec- trometry-based ABPP to establish binding profiles for compounds in our DUB-targeting library of small mole- cules. Aim 2. To develop a data-independent thermal proteome profiling (TPP-DIA) chemoproteomic platform for DUB-targeting compounds. We will develop thermal proteome profiling (TPP) assays which will directly quantify the DUBome or proteome-wide binding behavior of DUB-targeting small molecules in live cells. We will first utilize our new DUB-ABPs as enrichment reagents to generate mass spectral libraries for DUBs and Ub-l binding proteins. We will use these reference spectra as the foundation for data-independent acquisition (DIA) thermal profiling chemoproteomic assays at the DUBome (TPP-DIA-DUBome) and proteome-wide (TPP- DIA-Proteome) levels. Compounds from our DUB-focused library spanning a range of selectivity will be ana- lyzed on these new chemoproteomic platforms. Successful completion of our objectives will provide the field with a comprehensive and powerful set of rea- gents and technologies to rapidly develop hits, leads, and probes for DUBs, and further credential members of this important, but understudied protein family as novel drug targets in cancer.

摘要 泛素-蛋白酶体系统（UPS）在过去的15年中已经成为一个令人兴奋的新药物 癌症治疗的途径。100个去泛素化酶（DUBs）家族从亚细胞中去除泛素， 基质蛋白最近的研究表明，药物抑制DUBs可能提供了一个机会， 以改进的精确度治疗癌症中的癌蛋白。尽管越来越多的人对他们的功能感兴趣， 由于DUB作为治疗靶点的作用和潜力，研究DUB的试剂和分析平台有限。 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）是开发新的DUB聚焦活性试剂和相关的分析 方法.实现我们的里程碑将直接支持我们和其他人在近期（3-5年）的努力， 该领域开发选择性工具化合物，以询问基本DUB生物学并进一步验证特定DUB 作为抗癌药物的靶点。我们将通过两个具体目标来实现我们的目标： 目标1.开发和表征新型DUB基于活动的探针（ABP），用于基于活动的 蛋白质分析（DUB-ABPP）。我们将开发一个扩大的DUB-ABP集合，包括PTM- 修饰的泛素。我们将对每个ABP进行验证，以确认其选择性参与和丰富子集的能力 的蜂窝DUBs。符合这一里程碑的探针将在多路复用、质谱分析中组合使用。 基于比色法的ABPP，以建立我们的DUB靶向小分子化合物库中化合物的结合特征， 克里斯 目标2.建立一个与数据无关的热蛋白质组分析（TPP-DIA）平台 用于DUB靶向化合物。我们将开发热蛋白质组分析（TPP）检测， 定量DUB靶向小分子在活细胞中的DUBome或蛋白质组范围的结合行为。我们 将首先利用我们新的DUB-ABP作为富集试剂，生成DUB的质谱库， Ub-l结合蛋白。我们将使用这些参考光谱作为数据独立采集的基础 (DIA)DUBome（TPP-DIA-DUBome）和蛋白质组范围（TPP- DIA-蛋白质组）水平。化合物从我们的DUB为重点的图书馆跨越一系列的选择性将被分析， 在这些新的化学蛋白质组学平台上裂解。 成功完成我们的目标将为该领域提供一套全面而强大的现实， 快速开发DUB的命中率、线索和探测器，以及 这个重要的，但研究不足的蛋白质家族作为癌症的新药物靶点。