基于MDM2-p53和MDMX-p53蛋白-蛋白相互作用的双重抑制剂SY1108的结构优化及抗肿瘤活性研究

P53 is an important tumor suppressor gene, which plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 (murine double minute 2) and MDMX (murine double minute X) are both p53-binding proteins that regulate p53 stability and activity. Overexpression of MDM2 and MDMX abrogates the ability of p53 to induce cell cycle arrest and apoptosis. MDM2 and MDMX are key factors in tolerance of wild type p53 in tumors, making them attractive targets for the development of novel anti-tumor agents. Recovery of p53 function by disrupting the p53-MDM2 and p53-MDMX interactions using small-molecule antagonists could provide an efficient nongenotoxic anticancer therapy. In our previous studies, we discovered the β-lactam thiazepine compound SY1108 through ligand-based and structure-based virtual screening. The compound exhibited dual MDM2-p53 and MDMX-p53 inhibitory activities. In addition, SY1108 showed anti-tumor activity against HCT116, MCF7 and A549 cell lines in vitro, and inhibited the tumor growth in vivo, with anti-tumor activities comparable to that of reference drug nutlin-3a. In this project, SY1108 was chosen as the lead compound based on preliminary results. Six novel series was designed through CADD (computer aided drug design) methods, such as docking pose analysis and scaffold hopping. The evaluation of MDM2-p53 and MDMX-p53 inhibitory activities, anti-tumor activities, and mechanism studies of all the synthesized compounds will be carried out, to discover promising agents with potent anti-tumor activity and superior physicochemical and ADMET profiles.

p53是一种关键的抑癌基因。恶性肿瘤中高表达的MDM2及MDMX能够结合于p53蛋白，从而抑制其功能，增加肿瘤发生风险。设计小分子抑制剂阻断MDM2、MDMX与p53的相互作用是目前抗肿瘤药物的研发策略之一。课题前期以MDM2-p53和MDMX-p53蛋白-蛋白相互作用为靶点，通过虚拟筛选研究，发现β-内酰胺并硫氮杂䓬结构的化合物SY1108，该化合物具有MDM2-p53和MDMX-p53双重抑制活性，同时在体外及体内肿瘤模型上表现出抑制肿瘤生长的活性，未见此结构有相关活性的报道。本课题将在前期研究的基础上，以SY1108为先导化合物，借助于计算机辅助药物设计手段，设计六类全新骨架类型的化合物。对合成化合物通过荧光偏振实验进行靶点活性测试，进一步对其体外抗肿瘤活性及相关机制进行研究，最后通过活体成像技术评价其对肿瘤生长及转移的抑制情况。以期得到靶点明确、抗肿瘤活性更强、成药性好的抗肿瘤化合物。

恶性肿瘤严重威胁着人类的生命。p53基因是迄今为止已发现的与人肿瘤发生相关性最高的抑癌基因，MDM2及MDMX能与p53蛋白结合从而抑制p53的功能。阻断MDM2-p53及MDMX-p53的相互作用，激活p53，恢复其功能，是一种很有前途的恶性肿瘤治疗策略。本项目以SY1108为先导化合物进行结构改造，共设计合成目标化合物204个。采用荧光偏振法对目标化合物进行MDM2及MDMX亲和活性测试，共有15个化合物抑制活性优于阳性药Nutlin-3a，较优的化合物II-6和II-14，其Ki值分别为0.035和0.024 μM。采用MTT法对部分目标化合物进行抗肿瘤活性测试，多数测试化合物均表现出优于阳性药Nutlin-3a的活性，化合物I-6、II-11、II-12、II-14、II-15及VII-13抗肿瘤活性较好。通过Western实验验证化合物的作用机制，所测试化合物II-6、II-11、II-12、II-14、II-15及VII-13能够浓度依赖性的升高HCT116细胞内MDM2、MDMX及p53蛋白的含量，表明化合物能够干扰p53-MDM2及p53-MDMX蛋白-蛋白相互作用。化合物II-6、II-14及VII-13对细胞产生了明显的阻滞作用，使肿瘤细胞停滞了G1期。小鼠体内实验表明，化合物II-6、II-14及VII-13能够明显肿瘤的生长，对人结肠癌细胞小鼠模型抑瘤率分别为58.0%、43.7%、53.0%。本项目为开发新型的具有MDM2-p53和MDMX-p53双重抑制作用的抗肿瘤药物奠定的较好的基础。

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