The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR–Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days.

小分子降解剂（例如，靶向嵌合体的蛋白水解）的合理开发仍然是一个挑战，因为决定降解剂效率的限速步骤在很大程度上是未知的。靶向蛋白质降解领域的标准方法主要依赖于经典的低通量终点测定，例如蛋白质印迹或定量蛋白质组学。在这里，我们应用基于 NanoLuciferase 和 HaloTag 的筛选技术来确定感兴趣的蛋白质和选定的 E3 连接酶之间小分子诱导的三元复合物形成的动力学和稳定性。设计了一系列活细胞测定，以探测降解过程中最关键的步骤，同时最大限度地减少所需表达构建体的数量，使拟议的测定管道灵活且能够适应用户的要求。这种方法评估了选择性目标降解剂的潜在机制，并揭示了活细胞中开发的降解剂分子的确切特征。该方案允许受过基础细胞培养和分子生物学培训的科学家在建立后 2 周内通过跟踪三元复合物的形成来进行小分子邻近诱导物筛选，而使用 HiBiT 系统的降解剂筛选需要 CRISPR-Cas9 工程细胞系其生成可能需要长达 3 个月的时间。细胞系生成后，降解剂筛选和验证可以在几天内以高通量方式进行。