Piperlongumine (PL) conjugates induce targeted protein degradation

PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-Ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation. Overall design: To evaluate the anti-cancer efficacy and the specificity of 955, RNA-seq was performed to compare 955 with its warhead SNS-032 in MOLT4 cells.

蛋白降解靶向嵌合体（PROteolysis Targeting Chimeras, 简称PROTACs）是一类双功能分子，通过招募E3泛素连接酶（E3 ligases）实现靶蛋白的降解。然而其应用存在一定局限，部分原因在于目前已知的E3泛素连接酶配体可靶向招募的E3泛素连接酶种类十分有限。本研究中，我们鉴定出天然产物胡椒内酰胺（piperlongumine, PL）作为一种共价E3泛素连接酶招募因子：当其与细胞周期蛋白依赖性激酶9（CDK9）抑制剂SNS-032结合后，可有效诱导CDK9降解。先导化合物955能够以泛素-蛋白酶体依赖的方式高效降解CDK9，且在体外对多种肿瘤细胞的增殖抑制活性远强于SNS-032。机制层面的研究显示，我们通过基于TurboID的蛋白质组学实验，鉴定出Kelch样ECH相关蛋白1（KEAP1）为955所招募的、用于降解CDK9的E3泛素连接酶，该结论后续通过KEAP1基因敲除实验以及纳米生物发光共振能量转移（nanoBRET）三元复合物形成实验得到了验证。此外，PL-色瑞替尼（Ceritinib）结合物可降解棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶（EML4-ALK）融合癌蛋白，这一结果提示PL作为共价E3泛素连接酶配体，在靶向蛋白质降解领域具备更广泛的应用潜力。
总体实验设计：为评估955的抗癌活性与作用特异性，本研究在MOLT4细胞中开展RNA测序（RNA-seq）实验，将955与其弹头基团SNS-032进行对比分析。