Jones Hannah B L, Heilig Raphael, Fischer Roman, Kessler Benedikt M, Pinto-Fernández Adán
Nuffield Department of Medicine, Target Discovery Institute, Centre for Medicines Discovery, University of Oxford, Oxford, United Kingdom.
Front Chem. 2021 Feb 25;9:640105. doi: 10.3389/fchem.2021.640105. eCollection 2021.
The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.
小分子抑制剂的效力和选择性是药物发现早期阶段需要评估的关键参数。特别是，在相关细胞环境中表征化合物以揭示潜在的脱靶效应和药物疗效非常有意义。基于活性的探针是用于该目的的有价值工具，然而，以高通量形式获得细胞靶标结合数据一直特别具有挑战性。在此，我们描述了一种名为ABPP-HT（高通量兼容的基于活性的蛋白质谱分析）的新方法，该方法实施了一种半自动蛋白质组学样品制备工作流程，在保留其酶谱分析特征的同时，将经典ABPP工作流程的通量能力提高了约十倍。使用一组去泛素化酶（DUB）抑制剂，我们证明了ABPP-HT在细胞环境中提供内源性DUB的化合物选择性谱的可行性，与以前的方法相比，所需时间仅为其一小部分。
