Wang Changli, Liu Wanting, Guo Haotian, Lan Tian, Wang Tianyi, Wang Bing
Institute of Biochemistry and Molecular Biology, College of Life and Health Sciences, Northeastern University, Shenyang 110819, China.
Int J Mol Sci. 2025 May 30;26(11):5286. doi: 10.3390/ijms26115286.
Murine double minute 2 (MDM2) is involved in various cancers and is an attractive target. The RING domain of MDM2 has been discussed as an alternative target to stabilize p53. Designing drugs to target the RING domain of MDM2 is an alternative approach to preventing MDM2-mediated deactivation of p53. In this study, we obtained a human VH single-domain antibody and revealed its regulatory effects and mechanisms. The RING domain of MDM2 was synthesized using a chemical synthesis method, and antibodies against the MDM2 RING domain were screened from a human VH single-domain antibody library and expressed intracellularly. A nuclear localization sequence was designed to ensure intrabody efficiency. The binding activity of the individually cloned antibodies was detected using ELISA. MTT and flow cytometry assays were used to detect the reactions related to intrabody in vitro. The combination and its influence on MDM2 were detected using immunoprecipitation assays, confocal microscopy, and Western blotting. The effects on apoptosis-related mitochondrial pathways downstream of p53 were examined using Western blotting. The influence on cell cycle distribution and cyclin-related proteins was detected using flow cytometry and Western blotting. A549 cell xenografts were constructed to assess the effect of intrabodies on growth in vivo. The molecular mechanisms of MDM2 and p53 were studied using Western blotting. Eight individual cloned antibodies were positive compared to the signals on the BSA-coated plates, especially intrabodies VH-HT3. In A549 and MCF-7 cell lines, VH-HT3 exhibited significant inhibitory effects on cell proliferation and apoptosis. VH-HT3 co-localized with MDM2 in the nucleus and cytoplasm. The specific combination of VH-HT3 triggered no significant effect on MDM2 activity for p53 degradation but upregulated the levels of factors downstream of p53, especially those in the mitochondrial apoptosis pathway. Moreover, VH-HT3 induced cell cycle arrest, and the expression of cyclin-related proteins was consistent with this observation. VH-HT3 also retarded the growth of A549 xenografts in vivo. Further tests suggested that VH-HT3 inhibited MDM2 function by increasing HIPK2 levels and activating p53 at the Ser46 site. VH-HT3, prepared from a human VH single-domain antibody library, inhibited p53 activity and produced a tumor-suppressive effect. The intrabody VH-HT3 is a candidate for the development of novel MDM2 inhibitors.
小鼠双微体2（MDM2）与多种癌症相关，是一个有吸引力的靶点。MDM2的RING结构域已被讨论作为稳定p53的替代靶点。设计靶向MDM2的RING结构域的药物是防止MDM2介导的p53失活的一种替代方法。在本研究中，我们获得了一种人VH单域抗体，并揭示了其调节作用和机制。采用化学合成方法合成MDM2的RING结构域，并从人VH单域抗体库中筛选针对MDM2 RING结构域的抗体并在细胞内表达。设计核定位序列以确保胞内抗体的效率。使用ELISA检测各个克隆抗体的结合活性。采用MTT和流式细胞术检测体外与胞内抗体相关的反应。使用免疫沉淀试验、共聚焦显微镜和蛋白质印迹法检测与MDM2的结合及其影响。使用蛋白质印迹法检测对p53下游凋亡相关线粒体途径的影响。使用流式细胞术和蛋白质印迹法检测对细胞周期分布和细胞周期蛋白相关蛋白的影响。构建A549细胞异种移植模型以评估胞内抗体对体内生长的影响。使用蛋白质印迹法研究MDM2和p53的分子机制。与包被牛血清白蛋白的平板上的信号相比，8个单独克隆的抗体呈阳性，尤其是胞内抗体VH-HT3。在A549和MCF-7细胞系中，VH-HT3对细胞增殖和凋亡表现出显著的抑制作用。VH-HT3在细胞核和细胞质中与MDM2共定位。VH-HT3的特异性结合对MDM2介导的p53降解活性没有显著影响，但上调了p53下游因子的水平，尤其是线粒体凋亡途径中的因子。此外，VH-HT3诱导细胞周期停滞，细胞周期蛋白相关蛋白的表达与这一观察结果一致。VH-HT3在体内也抑制了A549异种移植瘤的生长。进一步的试验表明，VH-HT3通过增加HIPK2水平并在Ser46位点激活p53来抑制MDM2功能。从人VH单域抗体库制备的VH-HT3抑制p53活性并产生肿瘤抑制作用。胞内抗体VH-HT3是新型MDM2抑制剂开发的候选物。
