Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2010 Aug 10;107(32):14321-6. doi: 10.1073/pnas.1008930107. Epub 2010 Jul 26.
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed (D)PMI-alpha (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed (D)PMI-gamma (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both (D)PMI-alpha and (D)PMI-gamma failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, (D)PMI-alpha exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.
癌蛋白 MDM2 和 MDMX 负调控肿瘤抑制蛋白 p53 的活性和稳定性，赋予肿瘤发生和存活能力。针对 MDM2 和 MDMX 与 p53 结合域的拮抗剂通过重新激活 p53 通路，在体外和体内杀死肿瘤细胞，为癌症治疗提供了一类抗肿瘤药物。在天然化学连接和镜像噬菌体展示的辅助下，我们最近鉴定出一种 p53-MDM2 相互作用的 D-肽抑制剂，称为（D）PMI-alpha（TNWYANLEKLLR），其与 p53 竞争与 MDM2 的结合亲和力为 219 nM。增加选择严格性导致一种独特的 D-肽抑制剂，称为（D）PMI-gamma（DWWPLAFEALLR），其与 MDM2 的亲和力为 53 nM。结构研究与突变分析相结合，验证了这些 D-肽作为 MDM2 依赖性 p53 激活剂的作用模式。尽管对蛋白水解具有抗性，但（D）PMI-alpha 和（D）PMI-gamma 均无法主动穿过细胞膜，并且当与阳离子细胞穿透肽缀合时，无论 p53 状态如何，均具有非选择性细胞毒性。然而，当用整合素靶向环状-RGD 肽修饰的脂质体包封时，（D）PMI-alpha 在细胞培养物和裸鼠异种移植模型中对人胶质母细胞瘤发挥了强大的 p53 依赖性生长抑制活性。我们的研究结果验证了 MDM2 的 D-肽拮抗剂作为一类 p53 激活剂，可用于靶向治疗携带有 WT p53 和高水平 MDM2 的恶性肿瘤的分子靶向治疗。
