Fluorescence-Coupled Ubiquitination Assay as a High-Throughput Screening Strategy for Novel Cereblon Degraders

Cereblon (CRBN)-based protein degradation, via molecular glue degraders (MGDs) and proteolysis-targeting chimeras (PROTACs), is a promising cancer treatment strategy in targeted protein degradation (TPD). However, novel degraders discovery remains limited due to the lack of robust, high-throughput screening (HTS) methods for processing pools of purified compounds or complex chemical synthesis mixtures. Here, we introduce an innovative HTS strategy that employs a highly sensitive, fluorescence-coupled ubiquitination assay to identify CRBN-based degraders. This approach tracks ubiquitinated target proteins via gel-based analyses, and thereby progressively narrows down the list of potential degrader molecules from large-scale compound libraries or chemical reaction mixtures. Using this strategy, we identified LL-BPTF-8, a promising lead compound of PROTAC degrader with high potency and selectivity that targets the bromodomain PHD finger transcription factor (BPTF). Overall, our method offers a low-cost, rapid, and versatile platform for the HTS of protein degrader candidates, significantly streamlining the discovery of novel degraders.基于脑蛋白（Cereblon, CRBN）的蛋白降解策略，通过分子胶降解剂（molecular glue degraders, MGDs）与蛋白水解靶向嵌合体（proteolysis-targeting chimeras, PROTACs）实现，是靶向蛋白降解（targeted protein degradation, TPD）领域极具潜力的癌症治疗方案。然而，由于缺乏能够处理纯化化合物库或复杂化学合成混合物的稳健高通量筛选（high-throughput screening, HTS）方法，新型降解剂的发现仍受到诸多限制。本研究提出一种创新性HTS策略，该策略借助高灵敏度荧光偶联泛素化检测技术，用以识别基于CRBN的降解剂。该方法通过凝胶分析追踪泛素化靶蛋白，从而可从大规模化合物库或化学反应混合物中逐步缩小潜在降解剂分子的候选范围。利用该策略，我们成功鉴定出LL-BPTF-8——一种靶向溴结构域PHD指转录因子（bromodomain PHD finger transcription factor, BPTF）的高效且高选择性的PROTAC降解剂先导化合物。总体而言，本方法为蛋白降解剂候选物的HTS提供了低成本、快速且通用的技术平台，可显著简化新型降解剂的发现流程。