Maiwald Samuel, Heim Christopher, Hernandez Alvarez Birte, Hartmann Marcus D
Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany.
ACS Med Chem Lett. 2020 Dec 4;12(1):74-81. doi: 10.1021/acsmedchemlett.0c00440. eCollection 2021 Jan 14.
Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN).
通过定制分子胶或蛋白酶靶向嵌合体（PROTAC）将E3泛素连接酶重新用于靶向蛋白质降解是一种日益重要的治疗方式。目前，设计合适的分子胶和PROTAC的一个主要限制是我们对E3连接酶及其配体空间的了解不完整。我们在此描述了一种基于定制报告配体的热泳行为来发现和表征E3连接酶配体的定量分析方法。因此，它与常用的基于荧光的分析方法正交，并且受测试化合物光学性质的影响较小。它可用于针对给定连接酶的化合物库的高通量筛选，也可用于命中验证，我们通过鉴定意外的良好结合剂和非结合剂来证明这一点，从而对大脑神经酰胺（CRBN）的配体空间有了新的认识。
