Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, β-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells.

通过使用小分子强制靶标接近 E3 泛素连接酶而诱导的靶向蛋白降解 (TPD) 已成为针对以前无法成药的致病蛋白的重要药物发现方法。然而，在人类基因组编码的 600 多种 E3 连接酶中，目前只有 10 多种 E3 连接酶用于 TPD。在这里，使用亲和定向蛋白导弹 (AdPROM) 系统，其中抗 GFP 纳米抗体与 E3 连接酶连接，我们筛选了 30 多种 E3 连接酶，以确定它们降解 4 种目标蛋白（K-RAS、STK33、β）的能力。 -连环蛋白和 FoxP3，内源性 GFP 标记。几种新的 E3 连接酶，包括 CUL2 二甘氨酸受体 KLHDC2，作为有效的降解剂出现，表明这些 E3 连接酶可用于小分子降解剂的开发，例如蛋白水解靶向嵌合体 (PROTAC)。作为概念证明，我们证明连接到氯烷的基于 KLHDC2 募集肽的 PROTAC 能够降解细胞中的 HALO-GFP 蛋白。