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.

抽象的 泛素蛋白酶体系统 (UPS) 在过去 15 年中作为一种令人兴奋的新型药物而出现 癌症治疗的途径。 100 种去泛素化酶 (DUB) 家族可从亚基中去除泛素 策略蛋白。最近的研究表明，DUB 的药物抑制可能提供了一个机会 以更高的精度治疗癌症中的癌蛋白。尽管人们对其功能越来越感兴趣 由于DUBs作为治疗靶点的潜力和潜力，用于研究DUBs的试剂和分析平台有限。 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- ochemistry，化学蛋白质组学）致力于开发新型DUB活性试剂和相关分析 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 开发选择性工具化合物来探究基本 DUB 生物学并进一步验证特定 DUB 作为癌症药物靶点。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 活性探针 (ABP)，用于基于活性的探针 蛋白质分析（DUB-ABPP）。我们将开发更多 DUB-ABP 集合，包括 PTM- 修饰的泛素。我们将验证每个 ABP，以确认他们有选择性地参与和丰富子集的能力 蜂窝 DUB。达到这一里程碑的探针将在多重、质谱中组合使用 基于 trometry 的 ABPP 为我们的小分子 DUB 靶向库中的化合物建立结合谱 库勒斯。 目标 2. 开发数据独立的热蛋白质组分析 (TPP-DIA) 化学蛋白质组平台 用于 DUB 靶向化合物。我们将开发热蛋白质组分析（TPP）测定法，该测定法将直接 量化活细胞中 DUB 靶向小分子的 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 作为富集试剂来生成 DUB 的质谱库 Ub-1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) 和全蛋白质组 (TPP- DIA-蛋白质组）水平。我们以 DUB 为重点的库中涵盖一系列选择性的化合物将被分析 在这些新的化学蛋白质组平台上裂解。 成功完成我们的目标将为该领域提供一套全面而强大的目标 人员和技术，以快速开发 DUB 的热门歌曲、线索和探针，以及进一步的认证成员 这个重要但尚未得到充分研究的蛋白质家族作为癌症的新药物靶点。