新型USP7抑制剂的设计、合成与生物活性研究

The deubiquitinating enzyme USP7 is a biomarker of some tumors, and plays an important role in tumorigenesis and tumor development by enhancing the stability of key carcinogenic proteins such as MDM2 and DMNT1 by deubiquitination, and thus is regarded as a promising new anti-tumor target. However, the potency and selectivity of current USP7 inhibitors are not so good that by far there is not any inhibitor entering into clinical studies. In recent years, this applicant has been working on USP7 inhibitors. By analysis of crystallographic structures, we found that the ubiquitin C-terminal binding site (located at the catalytic domain) of USP7 was a drug-hot-binding pocket, and its residues such as Asp295 were also involved in the unique activation mechanism of USP7. We proposed that this site could be used to design novel and highly effective USP7 inhibitors. To this end, we conducted a virtual compound screening and activity test for the hits based on this site. As a result, three selective USP7 inhibitors were obtained. Based on this finding, this project will be focused on discovery of new lead compounds, structural modifications, biological evaluation, and mechanistic studies in order to find potent, selective and safe USP7 inhibitors. The project is expected to provide a breakthrough for the design of potent and highly selective USP7 inhibitors and to provide new avenues for cancer therapy and drug candidates.

去泛素化酶USP7是多种肿瘤的生物标志物，其通过去泛素化作用使MDM2、DMNT1等致癌蛋白的稳定性增强，因而在肿瘤发生发展过程中发挥重要作用，被认为是极具前景的抗肿瘤新靶标。文献报道的USP7抑制剂活性和选择性均不够理想，至今尚未有任何分子进入临床研究。申请人近几年一直从事新型USP7抑制剂研究，我们通过分析USP7-泛素的复合晶体结构，发现USP7的泛素碳未端结合位点（位于催化域）是药物热点结合口袋，其Asp295等残基还参与了USP7-C端所介导的酶活化机制，因而可用于设计高效新型USP7抑制剂。前期研究针对该位点进行了虚拟筛选和实体化合物活性测试，发现3个具有一定选择性的USP7抑制剂先导化合物。在此基础上，本项目拟进一步开展新型先导化合物的发现、结构优化、生物活性评价及作用机制研究，以期获得活性强、选择性高且安全性好的新型USP7抑制剂，为肿瘤治疗提供新途径和候选药物分子。

去泛素化酶USP7高表达于多种肿瘤中，通过去泛素化作用调节MDM2、DNMT1、FoxP3等关键靶蛋白的稳定性、核内外窜梭及信号传导等，因此与肿瘤的发生发展密切相关。USP7催化域的泛素结合区对USP7发挥去泛素化功能至关重要，其将泛素C端引导至USP7的催化中心水解。本项目针对USP7催化域的泛素C端结合口袋进行虚拟筛选，获得若干个微摩尔级的USP7抑制剂苗头化合物，并基于当前报道最多但肝微粒体代谢稳定性差的N-酰基哌啶醇类USP7抑制剂结构探索新型USP7抑制剂分子设计，结果发现N-苄基哌啶醇化合物A62具有很强的USP7抑制活性（KD = 78.3 nM、IC50 = 41 nM）。共晶结构解析显示，A62与USP7的结合方式同当前报道最活跃的N-酰基哌啶醇类USP7抑制剂有很大不同。A62的哌啶环几乎“直立”而不是“平伏”在USP7的口袋中，醇羟基通过平伏键而不是直立键与哌啶环相连，N-苄基向USP7蛋白表面伸展，甲酸甲酯片段几乎穿出口袋并与氨基酸His461产生新的作用。A62代表了一类新型USP7抑制剂，但代谢稳定性也差，因此我们对A62进行结构优化，设计合成了200多个目标化合物开展生物活性评价。结果显示，化合物LML-17-133 和LX-03-82具有很强的USP7抑制活性（IC50 = 73.6 nM和19.6 nM）和良好的体外人肝微粒体代谢稳定性（T1/2 = 264.3 min和142.5 min）。体内药代动力学研究表明， 二者可口服吸收，但口服生物利用度不理想，LML-17-133在大鼠上的口服生物利用度为10.9%，LML-17-133 和LX-03-82在小鼠上的口服生物利用度分别为2.3%和3.6%。体外抗肿瘤活性实验表明，LML-17-133 和LX-03-82对人急性淋巴白血病细胞RS4;11（IC50 = 27.2 nM）和人前列腺癌细胞LNCaP（IC50 = 25.4 nM 和11.7 nM）均具有很强的增殖抑制活性，但对HCT116、NB4、K562、HuH-7、MC38等细胞则不敏感。在小鼠皮下MC38结肠癌模型上，LML-17-133等化合物呈现显著的抗肿瘤免疫作用。进一步的结构优化和生物活性评价仍在进行中。本项目研究成果为后续研制first-in-class抗肿瘤新药奠定了基础。