Covalent Recruitment of NEDD4 for Targeted Protein Degradation: Rational Design of Small Molecular Degraders

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 EGFRL858R/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.

靶向蛋白质降解（Targeted protein degradation, TPD）已成为治疗多种疾病的极具潜力的治疗策略。然而，当前小分子降解剂主要依赖CRBN、VHL等有限的E3泛素连接酶种类，这极大限制了其应用范围。本研究报道，将2H-吖丙因（2H-azirine）化学官能团引入EGFRL858R/T790M/C797S抑制剂中，可诱导靶蛋白发生显著降解。蛋白质组学分析与功能验证证实，NEDD4型E3泛素连接酶通过结合C1286残基，被2H-吖丙因共价招募，从而促进靶蛋白降解。进一步研究表明，2H-吖丙因基团具有良好的通用性：当与多种配体缀合时，该基团可作为小分子降解剂，有效介导细胞周期蛋白依赖性激酶4（CDK4）、磷酸二酯酶5（PDE5）、布鲁顿酪氨酸激酶（BTK）以及溴结构域蛋白4（Brd4）的降解。更为重要的是，采用相同的蛋白配体骨架，本研究证实基于2H-吖丙因的探针可降解那些无法通过CRBN或VHL招募实现降解的耐药蛋白。该策略为开发靶向替代E3泛素连接酶的新型小分子降解剂提供了理性的设计思路。值得注意的是，这类降解剂在抑制癌细胞增殖方面的效果显著优于其母本激酶抑制剂。