Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy for treating various diseases. However, current small molecule degraders predominantly rely on a limited set of E3 ubiquitin ligases, such as CRBN and VHL, which restricts their applications. Here, we report that incorporation of the 2H-azirine chemical handle into the EGFR^{L858R/T790M/C797S} inhibitor induced remarkable degradation of the targeted protein. Proteomic profiling and functional validation confirmed that the NEDD4 E3 ligase was covalently recruited by 2H-azirine through engagement of C1286 residue, facilitating target degradation. Furthermore, the 2H-azirine moiety demonstrated versatility by acting as a small molecular degrader when conjugated to various ligands, effectively mediating the degradation of CDK4, PDE5, BTK and Brd4. More importantly, using the identical protein ligand scaffold, we demonstrated that the 2H-azirine based probe can degrade proteins resistant to degradation by CRBN or VHL recruitment. This approach provides a rational strategy for developing novel small molecular degraders that target alternative E3 ubiquitin ligases. Notably, these degraders significantly outperformed their parent kinase inhibitor in suppressing cancer cell growth.

靶向蛋白降解（TPD）已成为治疗各种疾病的一种有前景的策略。然而，目前的小分子降解剂主要依赖于有限的一组E3泛素连接酶，如CRBN和VHL，这限制了它们的应用范围。在这里，我们报告将2H-氮丙啶化学手柄引入EGFR<L858R/T790M/C797S>抑制剂中可以显著降解靶蛋白。蛋白质组学分析和功能验证确认NEDD4 E3连接酶通过与C1286残基的结合被共价募集，从而促进目标蛋白的降解。此外，2H-氮丙啶部分表现出多功能性，当与其他配体共轭时可以作为小分子降解剂有效介导CDK4、PDE5、BTK和Brd4的降解。更重要的是，使用相同的蛋白质配体支架，我们证明基于2H-氮丙啶的探针可以降解CRBN或VHL招募难以降解的蛋白。该方法为开发针对替代E3泛素连接酶的小分子降解剂提供了合理的策略。值得注意的是，这些降解剂在抑制癌细胞生长方面显著优于它们对应的母体激酶抑制剂。