Tools for DUB Drug Discovery

摘要 泛素-蛋白酶体系统(UPS)在过去的15年里已经成为一种令人兴奋的新药物 癌症治疗的途径。100个脱泛素酶家族(DUBS)从亚基中去除泛素 层状蛋白质。最近的研究表明，对DUBS的药物抑制可能提供了一个机会 以更高的精确度治疗癌症中的癌蛋白。尽管人们对他们的功能越来越感兴趣- 作为治疗靶点，研究DUBS的试剂和分析平台有限。 我们跨学科团队的目标(Sara Buhrlage，配音生物学，化学生物学；Jarrod Marto蛋白质双- 化学，化学蛋白质组学)是开发新型的双聚焦活性试剂和相关的分析 方法：研究方法。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以询问基本Dub生物学并进一步验证特定Dub的领域 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： 目的1.开发和表征用于基于活性的新型复制探针(ABPs) 蛋白质图谱(DUB-ABPP)。我们将开发更多的DUB-ABP集合，包括PTM- 修饰泛素。我们将验证每个ABP，以确认其选择性参与和丰富子集的能力 手机配音。符合这一里程碑的探测器将被组合使用在多路复用、质谱分析- 基于比例法的ABPP为我们的小分子靶向库中的化合物建立结合图谱- 克洛斯。 目的2.建立与数据无关的热蛋白质组图谱(TPP-DIA)化学蛋白质组分析平台 用于配音靶向化合物。我们将开发热蛋白质组图谱(TPP)分析，它将直接 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 将首先利用我们的新DUB-ABPs作为富集剂来生成DUBS和DUB的质谱库 UB-L结合蛋白。我们将使用这些参考光谱作为独立于数据采集的基础 (DIA)热分析DUBome(TPP-DIA-DUBome)和全蛋白质组(TPP-DIA-DUBome) Dia-蛋白质组)水平。来自我们专注于DUB的库中的各种选择性化合物将被分析- 裂解在这些新的化学蛋白质组平台上。 成功完成我们的目标将为外地提供一套全面而强大的理性-- 绅士和技术，以快速开发命中、线索和探测，以获得配对和更多凭据成员 这一重要但未被充分研究的蛋白质家族是治疗癌症的新药物靶点。

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.