We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

我们描述了一种基于时间分辨福斯特共振能量转移 (TR-FRET) 的通用平台，用于分析异双功能降解剂（或蛋白水解靶向嵌合体 [PROTAC]）的细胞作用，该平台能够准确定量全细胞中的蛋白质水平在不到 1 小时内裂解产物并测量小分子靶标与内源蛋白的结合，此处专门针对人类含溴结构域蛋白 4 (BRD4)。检测混合物由针对目标蛋白质的单一一抗、发光供体标记的抗物种纳米抗体和荧光受体配体组成。重要的是，我们的策略可以很容易地应用于其他感兴趣的靶标，并将极大地促进使用未修饰的细胞系以高通量形式对小分子抑制剂和 PROTAC 进行基于细胞的分析。我们进一步验证了我们的平台在雷公藤红素（一种含对醌甲基化物的五环三萜类化合物）的表征方面验证了我们的平台，作为一种广泛的半胱氨酸靶向 E3 泛素连接酶弹头，可实现有效且高效的靶向蛋白质降解。