Ji Xiaojun, Miao Lei, Wu Yebin, Zhao Tingli, Si Yaxuan, Tan Xiaoyun, Zhou Qiuhua, Zuo Rui, Pei Junjie, Wu Jian, Ma Changyou, Ma Zhongjun, Xu Dan
Innovation Department of the Research Institute, Nanjing Chia-Tai Tianqing Pharmaceutical Co. Ltd., Nanjing 210046, PR China.
Department of Pharmacology, Nanjing Chia-Tai Tianqing Pharmaceutical Co. Ltd., Nanjing 210046, PR China.
Biol Methods Protoc. 2024 Mar 5;9(1):bpae014. doi: 10.1093/biomethods/bpae014. eCollection 2024.
Proteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.
蛋白酶靶向嵌合体（PROTAC）是一种蛋白质降解技术，近年来在新药开发中得到越来越广泛的应用。Akt是一种经典的丝氨酸/苏氨酸激酶，其激酶以外的作用近年来逐渐受到关注，使其成为PROTAC靶向的蛋白质之一。目前，有许多方法用于评估细胞内蛋白质降解，但每种方法都有其自身的优缺点。本研究旨在探讨使用纳米荧光素酶方法评估PROTAC（MS21和MS170）对细胞中泛Akt蛋白降解的可行性。在对该方法与经典的蛋白质印迹分析在各种细胞中进行全面比较，并测试多批次实验的稳定性后，我们发现纳米荧光素酶是一种高度准确、稳定、低成本且易于操作的方法，用于评估PROTAC对细胞内泛Akt的降解，具有短周期时间和高细胞扩展性。鉴于该方法的众多优点，推测它可扩展用于评估更多PROTAC靶蛋白的降解。总之，纳米荧光素酶是一种适用于PROTAC化合物早期蛋白质降解筛选的方法。
