新颖USP7小分子抑制剂Parthenolide的抗肿瘤活性和作用机制研究

Deubiquitinase USP7 is a celebrated component of Ubiquitin-Proteasome System. Our recent work revealed that USP7 played a positive role in Wnt pathway via stabilizing β-catenin and thus promoted the colorectal cancer development, indicating that USP7 could be a potent target for development of Wnt inhibitors. Here, for the first time, we discovered Parthenolide (PTL), a sesquiterpene lactone, as a novel inhibitor of USP7 activity via high throughput screening. Futhter study suggested that PTL could effectively antagonize the activity of Wnt signaling and thus inhibited the proliferation of colorectal cancer cells. In this project, with molecular cell biology and pharmacology techniques, the inhibitory activity, molecular mechanisms and pharmacophore of PTL as USP7 inhibitor will be deeply elucidated. We will further investigate molecular mechanisms by which PTL regulating Wnt signaling and perform in vitro and in vivo comprehensive pharmacological evaluation on the inhibitory activity of PTL on colorectal cancer. The role of USP7/Wnt in the anti-cancer activity of PTL will be analyzed as well. Completion of this project will illuminate the cellular target and mechanisms of PTL’s anti-tumor activity and discover USP7 inhibitors with novel chemical structure, which is of important meaning for developing anti-tumor drugs targeting USP7 to cure tumors with desregulated Wnt signaling.

去泛素化酶USP7是泛素-蛋白酶体系统的重要成员，我们前期研究表明USP7通过稳定β-catenin正向调控Wnt信号通路，与结直肠肿瘤发生发展密切相关，是该信号通路抑制剂药物研究的新颖分子靶点。基于天然产物库的高通量活性筛选，我们首次发现倍半萜类化合物小白菊内酯(Parthenolide, PTL)能够显著抑制USP7活性，进一步的研究表明PTL有效抑制Wnt信号通路以及结直肠肿瘤细胞增殖。本项目拟利用分子细胞生物学和药理学手段，深入研究PTL作为新颖USP7抑制剂的活性、机制及药效基团，探讨PTL调控Wnt通路的分子机制，系统评价PTL抑制结直肠肿瘤的活性并探讨USP7/Wnt在PTL发挥抗肿瘤活性中的作用。项目研究将解析PTL的抗肿瘤作用靶点和机制，发现新颖化学结构类型的USP7抑制剂，研究结果对于发现靶向USP7治疗Wnt信号通路过渡激活的肿瘤药物具有重要意义。

泛素特异蛋白酶USP7作为泛素-蛋白酶体系统的重要成员，可通过调控包括Wnt信号通路在内的多种信号通路促进肿瘤的发生发展。因此新颖USP7抑制剂的发现对于抗肿瘤药物研发和肿瘤治疗具有重要的意义。本课题对前期基于高通量活性筛选发现的新颖USP7小分子抑制剂倍半萜类化合物小白菊内酯(Parthenolide, PTL)的抗肿瘤活性和作用机制进行了深入探讨。Ub-Rho110/Ub-AMC荧光实验、Ub-VME/Ub-PA分子探针实验以及Di-Ub实验的结果表明小白菊内酯能够选择性抑制USP7活性；细胞热迁移实验、表面等离子共振和质谱实验结果进一步揭示小白菊内酯能够与USP7蛋白直接结合。进一步研究发现小白菊内酯能够通过抑制USP7活性提高Wnt信号通路关键转录因子β-连环蛋白的泛素化水平并加速其通过蛋白酶体降解。双荧光素酶报告基因体系检测发现小白菊内酯对Wnt3a诱导的Wnt信号通路活性和结直肠肿瘤细胞内异常激活的Wnt信号通路活性均有显著的抑制作用。蛋白免疫印迹和qRT-PCR实验同样发现小白菊内酯显著下调结直肠肿瘤细胞中Wnt信号通路下游靶基因的表达。另外，体外MTS、流式分析和蛋白免疫印迹结果显示小白菊内酯能够抑制结直肠肿瘤细胞增殖并诱导其凋亡，体内结果表明小白菊内酯对HCT116裸鼠移植瘤的生长也有一定抑制作用。在此基础上，对另外两个倍半萜类化合物costunolide和α-santonin抑制USP7与Wnt信号通路的活性分析表明α-亚甲基-γ-丁内酯是倍半萜类化合物抑制USP7的潜在活性基团。综上，本项目的实施丰富了小白菊内酯抗结直肠肿瘤的分子机制，并为新颖结构类型USP7小分子抑制剂的研发提供了重要的基础实验数据。