靶向NAC1同源二聚体小分子抑制剂NIC19鉴定及其与化疗药物协同抗肿瘤作用研究  

Identification and antitumor effect of a small molecule compound NIC19 targeting the NAC1 homodimerization

作　　者：邹建华 董顺利 张晶晶 张熠[2] ZOU Jian-hua;DONG Shun-li;ZHANG Jing-jing;ZAHNG Yi(Department of Pharmacy,Peoples Hospital of SND Suzhou 215000,P.R.China;College of Pharmaceutical Sciences Medical College of SooChow University,Suzhou 215123,P.R.China)

机构地区：[1]苏州市高新区人民医院药剂科,江苏苏州215000 [2]苏州大学医学部药学院,江苏苏州215123

出　　处：《中华肿瘤防治杂志》2020年第3期179-186,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81773749;81473240)。

摘　　要：目的致癌转录因子的过度激活与肿瘤无限增殖、侵袭转移、耐药等恶性特征密切相关,靶向致癌转录因子为主要策略的肿瘤治疗日益受到关注。本课题基于NAC1蛋白二聚体结构进行了虚拟筛选,找到了另一个小分子先导化合物NIC19,并探究其药理作用及临床潜在意义。方法计算机辅助药物筛选发现靶向NAC1蛋白同源二聚体化合物NIC19;蛋白质印迹法检测NIC19的降解和表达;四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法和克隆集落形成实验检测NIC19对抗肿瘤药物作用的影响;裸鼠皮下肿瘤模型进一步验证NIC19对抗肿瘤药物作用。结果NIC19的发现及计算机辅助NIC19与NAC1蛋白对接模型;NIC19可下调NAC1蛋白的表达和稳定性,呈现时间和剂量依赖性(时间:与0μmol/L表达量2.62±0.1相比,NAC1蛋白的表达量降至5μmol/L的1.85±0.13,10μmol/L的1.18±0.03,20μmol/L的0.65±0.03,差异有统计学意义,F=63.703,P<0.001;剂量:与0h表达量2.43±0.13相比,NAC1蛋白表达量降至12h的2.30±0.03,24h的2.02±0.04,48h的0.49±0.15,差异有统计学意义,H=10.385,P=0.016);NIC19可增加肿瘤细胞对顺铂(cisplatin,DDP)或多柔比星(doxorubicin,DOX)治疗的敏感性(与DOX组相比t=9.220,P=0.0002;与DDP组相比t=6.844,P=0.001)。同时,克隆集落形成实验表明,联合NIC19可增强在DOX和DDP治疗下抑制SKOV3克隆集落形成能力(FNIC19×DOX=16.472,P<0.001;FNIC19×DDP=15.412,P<0.001),并且DDP或DOX与NIC19联合使用时,SKOV3凋亡水平明增高,表现为凋亡蛋白cleaved-PARP水平升高(FNIC19×DDP=7.041,P=0.029;FNIC19×DOX=6.098,P=0.039)。在体研究进一步证实了NIC19可增强肿瘤细胞对DDP的敏感性,在皮下接种HeLa细胞的裸鼠中,NIC19和DDP联合使用可抑制移植瘤的生长,FNIC19×DDP=9.720,P=0.014。与NIC19或DDP单独使用组相比,联合用药组可增加瘤体凋亡的发生,表现为TUNEL染色阳性细胞数增加(H=10.421,P=0.015),增殖标志物Ki-67阳性率减少,H=9.425,P=0OBJECTIVE Excessive activation of oncogenic transcription factors is closely associated with malignant phenotypes,including proliferation,invasion,metastasis and therapeutic resistance,which attract a great deal of interests in this regard.This topic was based on the virtual screening of NAC1 protein dimer structure,and found another small molecule lead compound NIC19,and explored its pharmacological effects and clinical potential.METHODS Computer-assisted drug screening was performed to identify NAC1 protein homodimeric compound NIC19;Western boltting was used to detect the degradation and expression of NIC19;MTT and colony colony formation assays were used to detect the effect of NIC19 on antitumor drugs;subcutaneous mouse tumor model further verify the effect of NIC19 on anti-tumor drugs.RESULTS The discovery of NIC19 and the computer-aided docking model of NIC19 and NAC1 protein;NIC19 could downregulate the expression and stability of NAC1 protein in a time-and dose-dependent manner.time:compared with 0μmol/L expression 2.62±0.1,the expression of NAC1 protein decreased to 1.85±0.13 of 5μmol/L,1.18±0.03 of 10μmol/L.The difference was statistically significant(F=63.703,P<0.001).There was significant difference between 20μmol/L and 0.65±0.03(P<0.001).Dose:compared with 0 h,the expression of NAC1 protein decreased to 2.30±0.03 at 12 h,2.02±0.04 at 24 h,and 0.49±0.15 at 48 h.NIC19 could increase the sensitivity of tumor cells to cisplatin(DDP)or doxorubicin(DOX)(compared with DOX group:t=9.220,P=0.0002;compared with DDP group:t=6.844,P=0.001).At the same time,the colony formation experiment showed that the combination of NIC19 could enhance the ability to inhibit the colony formation of SKOV3 under the treatment of DOX and DDP(FNIC19×DOX=16.472,P<0.001;FNIC19×DDP=15.412,P<0.001).The level of apoptosis in SKOV3 treated with DDP or DOX combined with NIC19 was significantly increased,showing that the level of apoptotic protein cleaved-PARP was increased(FNIC19×DDP=7.041,P=0.029;FNIC19×DOX=6.098,P=0.039

关 键 词：NAC1 二聚体 抑制剂 联合治疗 

分 类 号：R730.5[医药卫生—肿瘤]