Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, United States.
Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, United States.
Methods Enzymol. 2023;681:189-214. doi: 10.1016/bs.mie.2022.11.001. Epub 2022 Dec 19.
In recent years, Proteolysis Targeting Chimera (PROTAC) technology has emerged as one of the most promising approaches to remove disease-associated proteins by utilizing cells' own destruction machinery. To achieve successful degradation of a protein of interest (POI), the heterobifunctional PROTAC molecules must penetrate into the cells first, followed by target engagement and formation of the POI-PROTAC-E3 ligase complex. Based on this understanding, the assessment of cell permeability and in cell target engagement are of great importance to evaluate the efficacy of PROTAC candidates. PROTAC molecules can be classified as non-covalent and covalent, and covalent PROTACs can be further divided into irreversible and reversible covalent. Here, we present a high-throughput assay to prioritize different types of BTK PROTACs by measuring their intracellular accumulation quantitatively, using kinase binding assays and the NanoBRET target engagement platform.
近年来，蛋白水解靶向嵌合体（PROTAC）技术作为一种最有前途的方法之一，通过利用细胞自身的破坏机制来去除与疾病相关的蛋白质。为了成功降解靶蛋白（POI），必须首先使双功能 PROTAC 分子穿透细胞，然后与靶标结合并形成 POI-PROTAC-E3 连接酶复合物。基于这一认识，评估细胞通透性和细胞内靶标结合对于评估 PROTAC 候选物的疗效非常重要。PROTAC 分子可分为非共价和共价，共价 PROTAC 可进一步分为不可逆和可逆共价。在这里，我们提出了一种高通量测定法，通过使用激酶结合测定法和 NanoBRET 靶标结合平台，定量测量其细胞内积累，从而优先考虑不同类型的 BTK PROTAC。
