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Abstract：
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.
摘要：
蛋白水解靶向嵌合体(PROTAC)是近年来越来越多地应用于新药开发的一种蛋白质降解技术。Akt是一种经典的丝氨酸/苏氨酸激酶，其在激酶之外的作用近年来逐渐受到关注,使其成为PROTACs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，但是每个人都有自己的优点或缺点。本研究旨在探讨使用NanoLuc荧光素酶方法评估PROTACs（MS21和MS170）在细胞中降解pan-Akt蛋白的可行性。在对各种细胞中的这种方法与经典的蛋白质印迹测定法进行了彻底的比较后，以及测试多个批次之间实验的稳定性，我们发现NanoLuc荧光素酶是一种高度精确的，稳定,低成本且易于操作的方法，用于通过短周期时间和高细胞可扩展性的PROTACs评估细胞内泛Akt降解。鉴于此方法的众多优点，假设它可以扩展到评估PROTACs更多靶蛋白的降解。总之,NanoLuc荧光素酶是PROTAC化合物早期蛋白质降解筛选的合适方法。
