Structure-activity studies of Mdm2/Mdm4-binding stapled peptides comprising non-natural amino acids

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6–125) at 1.66 Å resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

作为p53的主要拮抗剂，鼠双微体2（Mdm2）及其近缘同源物鼠双微体4（Mdm4）是重要的癌症治疗靶点。我们此前已报道了一系列可穿透细胞的钉合肽（stapled peptides），这类肽可高亲和力结合Mdm2，进而激活p53肿瘤抑制因子（p53 tumour suppressor）。在该系列肽中，将保守色氨酸残基修饰为非天然L-6-氯色氨酸（L-6-chlorotryptophan）可获得最高的结合亲和力。为阐明亲和力提升的结构基础，我们解析了该钉合肽（M011）与Mdm2（6-125位残基）结合的晶体结构，分辨率达1.66埃（Å）。令人意外的是，该结构与未引入6-氯修饰的同源肽（M06）的结构几乎完全一致。对含有6-甲基色氨酸（6-Me-tryptophan）的线性肽与钉合肽的进一步分析，为同时拮抗Mdm2与Mdm4提供了机制层面的见解，并证实Mdm4的L98位点可作为可调控的空间位阻门控位点。本研究结果还揭示了柔性铰链区在决定Mdm2与Mdm4结构可塑性方面的潜在作用。