化合物C19抑制谷氨酰胺酶C活性研究

Validation of compound C19 as a glutaminase C inhibitor

线粒体中的谷氨酰胺酶C(GAC)在多种肿瘤中高表达,导致谷氨酰胺代谢增强而促进癌症发生发展,因此成为具有巨大潜力的抗肿瘤药物研发靶点。目前已发现的GAC抑制剂结构特征较为集中,鲜有全新结构骨架的抑制剂报道。本文构建了大肠杆菌原核表达体系,通过溶菌酶与超声方法裂解菌体,结合钴离子磁珠分离纯化技术得到了高纯度的人源GAC蛋白。在此基础上,建立热漂移检测、药物亲和反应的靶点稳定性分析、蛋白质交联技术和GAC酶活性检测方法,完善小分子与GAC蛋白的相互作用验证体系。利用这一体系,对最新报道自Enamine数据库中筛选到的化合物C19进行全面的分子-蛋白间相互作用确证和体外药效学研究。实验结果表明,C19直接与GAC蛋白相互作用,干扰GAC活性四聚体形成,进而降低其酶催化活性。通过抑制GAC的生物学功能,C19剂量依赖性地降低GAC介导的谷氨酰胺代谢,减少肿瘤细胞中GAC酶催化产物谷氨酸的含量,进而显著抑制A549和NCI-H1299非小细胞肺癌细胞增殖。综上,C19可作为先导化合物,对后续靶向GAC药物的结构设计提供新的思路。

The mitochondrial enzyme glutaminase C(GAC)is highly expressed in a variety of cancer cells,resulting in increased glutamine metabolism and cancer development.Therefore,GAC has become a potential target for anti-tumor drug development.However,current GAC inhibitors shared similar structural characteristics,few new scaffolds were reported.By conducting a prokaryotic Escherichia coli expression system,human GAC protein of high-purity was obtained through lysozyme digestion combined with ultrasound dissociation,and cobalt magnetic beads purification,Moreover,we performed studies to validate interaction between small molecules and GAC protein through thermal shift assay,drug affinity responsive target stability assay,protein crosslinking and GAC enzyme activity detection.Meanwhile,a comprehensive small molecule-protein interaction confirmation and systematic pharmacodynamic study in vitro were carried out on compound C19,which was a reported GAC inhibitor screened from the Enamine database.Results showed that C19 directly bind to GAC protein,disturbed GAC tetramers formation,and inhibited its enzyme catalytic activity.By interfering GAC function,C19 dose-dependently suppressed GAC-mediated glutamine metabolism,reduced glutamate in cancer cells,and thus alleviated A549 and NCI-H1299 non-small cell lung cancer cell growth.Together,C19 was identified as a lead compound,providing a new strategy for the structural design of drugs targeting GAC.