靶向CD147的小分子抑制剂的筛选及抗肿瘤机制研究
发布时间：2019-01-20 08:38
【摘要】：恶性肿瘤是世界范围内主要致死性疾病之一,亦是严重威胁人类生命健康的常见疾病[1]。恶性肿瘤异常增殖、抵抗凋亡以及侵袭转移等生物学特性,给肿瘤治疗带来了巨大挑战。传统的治疗手段尚无法从根本上治疗恶性肿瘤,且多伴随明显毒副作用,给患者带来痛苦,影响患者生活质量。近年来,分子靶向治疗作为肿瘤治疗的新策略,以其特异性强,副作用小等治疗优势逐渐被推崇,许多分子靶向药物已获FDA批准投入临床,并取得了较好的临床疗效。分子靶向治疗策略的核心是靶点的确定,通过对肿瘤分子机制不断的深入研究,寻找精准靶点,是攻克肿瘤的关键所在。免疫球蛋白超家族成员CD147分子是近年来报道较多的肿瘤相关分子(Tumor associated molecule,TAM)。研究表明,CD147分子在近20种不同来源的恶性肿瘤中呈现异常表达,并且通过不同的形式参与肿瘤发生,增殖,分化,侵袭转移,凋亡抵抗以及代谢等多种生物学事件,而通过干涉、敲除、阻断等方式抑制CD147的异常表达后,可以逆转肿瘤的恶性表型[2]。随着研究的深入,又成功解析了CD147分子的晶体结构,从而使基于分子结构的特异性药物筛选成为可能。可以说CD147已经具备了肿瘤的药物靶点特性,然而目前尚无靶向CD147的小分子药物。综上所述,本课题旨在基于CD147的分子结构,设计筛选出能够靶向CD147的抗肿瘤小分子抑制剂,并对其分子机制进行深入探讨。本课题主要包括三部分工作:第一部分,靶向CD147的小分子化合物的虚拟筛选及先导化合物AC-73的确定。我们应用计算机辅助药物筛选平台,以CD147的三维晶体结构为模型进行药效团模型的建立。随后对来自SPECS小分子化合物库中的近30万小分子化合物进行虚拟筛选和评分,最终得到综合排名靠前的100个候选小分子化合物。进一步通过SPR试验以及明胶酶谱试验对100个化合物进行初筛,确定了一个先导化合物AC-73。最后我们通过SPR试验,分子对接模拟技术以及点突变等试验预测AC-73与CD147结合时,可能存在的结合位点为CD147胞外段N端GLU64以及GLU73位氨基酸残基。第二部分,小分子化合物AC-73阻抑肝癌侵袭转移及分子机制研究。这一部分研究中,我们用CD147参与的恶性肿瘤中报道较多的肝细胞癌为研究模型,探讨AC-73的功能及分子机制。应用细胞划痕试验和侵袭小室试验,发现AC-73能够在体外抑制肝癌细胞迁移运动和侵袭转移,且该运动和侵袭能力的降低并非由AC-73影响了细胞活性所致。进一步通过比较AC-73在野生型肝癌细胞系和特异性敲除CD147的肝癌细胞系的药物活性的差异,证实AC-73抑制肝癌细胞运动和转移的能力是通过特异性靶向CD147实现的。应用Native SDS-PAGE/Western blot、明胶酶谱、RT-q PCR、蛋白免疫印迹等试验,对AC-73分子机制进行研究,结果表明AC-73可以通过抑制CD147同源二聚体形成的方式,下调CD147/ERK1/2/STAT3/MMP-2分子信号通路,从而抑制肝癌细胞发生侵袭转移。AC-73的上述功能特性和分子信号机制,在裸鼠肝原位移植瘤模型内进行了验证,并得到了证实。体内毒性试验显示,AC-73在小鼠体内耐受性较好,毒副作用小,具有良好的成药潜能。第三部分,AC-73改造化合物HA-08的抑瘤特性及促凋亡机制研究。在这一部分探索性工作中,我们对AC-73进行了结构改造,初期获得了八个改造化合物。经过分子对接模拟以及SPR试验初筛,得到一个能够与CD147相互作用的小分子化合物HA-08。对该小分子化合物进行了合成及纯化,用于后期的功能研究。侵袭小室试验证明,HA-08可以抑制肝癌细胞发生侵袭转移,同时发现HA-08对肝癌细胞活性产生了影响。Native SDS-PAGE/Western blot试验证实HA-08不能够抑制CD147形成同源二聚,提示HA-08的分子机制与AC-73存在差异。随后,我们应用WST-1试验检测了HA-08对9种不同肿瘤细胞活性影响,发现HA-08对肿瘤细胞具有较普遍的抑瘤活性。通过Annexin V-FITC/PI流式检测、JC-1和Hoechst染色以及Western blot等试验,发现HA-08能够促进肿瘤细胞发生凋亡,主要体现为诱导凋亡表型的出现以及凋亡相关蛋白水平的变化。HA-08的抑瘤特性以及促凋亡能力在荷瘤裸鼠模型上得到了证实。最后我们通过比较HA-08在CD147表达水平不同的肿瘤细胞的敏感性差异,明确HA-08的抑瘤活性和促肿瘤细胞凋亡的能力与CD147呈现显著相关性。
[Abstract]:Malignant tumor is one of the major fatal diseases in the world, and is a serious disease that seriously threatens the health of human life[1]. The abnormal proliferation of the malignant tumor, the resistance to apoptosis and the invasion and metastasis have brought great challenges to the treatment of the tumor. The traditional treatment method is not capable of fundamentally treating the malignant tumor, has the obvious toxic and side effect, brings pain to the patient, and influences the quality of the life of the patient. In recent years, molecular targeted therapy has been widely regarded as a new strategy for tumor treatment, with its specificity and small side effect, many of which have been approved by the FDA and have achieved good clinical curative effect. The core of the molecular targeted therapy strategy is the determination of the target, and it is the key to conquer the tumor by further studying the molecular mechanism of the tumor and finding the precise target. The CD147 molecule of the immunoglobulin superfamily has been reported in recent years with more tumor-related molecules (TAM). The study shows that CD147 is an abnormal expression in nearly 20 different kinds of malignant tumors, and is involved in various biological events such as tumorigenesis, proliferation, differentiation, invasion and metastasis, apoptosis resistance and metabolism through different forms, and by intervention and knockout, It is possible to reverse the malignant phenotype of the tumor after the suppression of the abnormal expression of the CD147 by blocking or the like[2]. With the development of the study, the crystal structure of the CD147 molecule is successfully resolved, and the specific drug screening based on the molecular structure can be made possible. CD147 can be said to have the drug target characteristic of the tumor, yet there is no small molecular medicine to target CD147 at present. To sum up, the purpose of this study is to design and screen anti-tumor micromolecule inhibitors which can target CD147 based on the molecular structure of CD147, and to probe into the molecular mechanism of CD147. This topic mainly includes three parts: the first part, the virtual screening of small molecular compound targeting CD147 and the determination of the pilot compound AC-73. We applied the computer-aided drug screening platform to establish the model of the pharmacophore model based on the three-dimensional crystal structure of CD147. and then performing virtual screening and scoring on the nearly 300,000 small molecular compound from the small molecular compound library of the SPECS, and finally obtaining the 100 candidate small molecule compounds in the comprehensive ranking. A pilot compound AC-73 was further determined by the SPR test and the gelatin enzyme spectrum test for 100 compounds. Finally, when the combination of AC-73 and CD147 is predicted by SPR test, molecular docking simulation technique and point mutation test, the possible binding site is the N-terminal GLUT64 and the GLU73 amino acid residue of the extracellular section of CD147. The second part, small molecule compound AC-73, represses the invasion and metastasis of the liver cancer and the molecular mechanism. In this part of the study, we used CD147 to report more hepatocellular carcinoma as the study model and to explore the function and molecular mechanism of AC-73. Using cell scratch test and invasive cell test, it was found that AC-73 was able to inhibit the migration and invasion of liver cancer cells in vitro, and the reduction of the movement and invasion ability was not caused by AC-73. Further, by comparing the differences in the drug activity of the AC-73 in the wild-type liver cancer cell line and the liver cancer cell line that specifically knocked out the CD147, it is confirmed that the ability of the AC-73 to inhibit the movement and metastasis of the liver cancer cells is achieved by the specific targeting of the CD147. The molecular mechanism of AC-73 was studied by means of Native SDS-PAGE, Western blot, Gelatinase, RT-q PCR and Western blot. The results showed that AC-73 could downregulate the signal pathway of CD147/ ERK1/ 2/ STAT3/ MMP-2 by inhibiting the formation of the CD147 homodimer, thereby inhibiting the invasion and metastasis of the liver cancer cells. The above-mentioned functional characteristics and molecular signal mechanism of AC-73 were verified in nude mouse liver in situ tumor model and confirmed. The in-vivo toxicity test shows that AC-73 is well tolerated in mice, has less toxic and side effect, and has a good biological potential. In the third part, the tumor-inhibiting characteristics of HA-08 and the mechanism of pro-apoptotic mechanism of the modified compound HA-08 were studied. In this part of the exploratory work, we carried out a structural transformation of AC-73 and initially obtained eight modified compounds. A small molecular compound HA-08 capable of interacting with the CD147 was obtained through the molecular docking simulation and the initial screen of the SPR test. The small molecular compound was synthesized and purified for later functional research. The invasion cell test demonstrated that HA-08 could inhibit the invasion and metastasis of the liver cancer cells, and also found that HA-08 has an effect on the activity of the liver cancer cells. Native SDS-PAGE/ Western blot proved that HA-08 could not inhibit CD147 formation of homolog, suggesting that the molecular mechanism of HA-08 was different from that of AC-73. Subsequently, we used WST-1 to test the effect of HA-08 on the activity of 9 different tumor cells, and found that HA-08 has a more common tumor-inhibiting activity on tumor cells. It was found that HA-08 could promote the apoptosis of tumor cells by Annexin V-FITC/ PI flow detection, JC-1 and Hoechst staining, and Western blot. The tumor-inhibiting characteristics and the pro-apoptotic ability of HA-08 were confirmed in the model of tumor-bearing nude mice. Finally, by comparing the sensitivity of HA-08 to tumor cells with different levels of CD147 expression, it is clear that the tumor-inhibiting activity of HA-08 and the ability to promote the apoptosis of tumor cells have a significant correlation with CD147.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5
本文编号：2411859
[Abstract]:Malignant tumor is one of the major fatal diseases in the world, and is a serious disease that seriously threatens the health of human life[1]. The abnormal proliferation of the malignant tumor, the resistance to apoptosis and the invasion and metastasis have brought great challenges to the treatment of the tumor. The traditional treatment method is not capable of fundamentally treating the malignant tumor, has the obvious toxic and side effect, brings pain to the patient, and influences the quality of the life of the patient. In recent years, molecular targeted therapy has been widely regarded as a new strategy for tumor treatment, with its specificity and small side effect, many of which have been approved by the FDA and have achieved good clinical curative effect. The core of the molecular targeted therapy strategy is the determination of the target, and it is the key to conquer the tumor by further studying the molecular mechanism of the tumor and finding the precise target. The CD147 molecule of the immunoglobulin superfamily has been reported in recent years with more tumor-related molecules (TAM). The study shows that CD147 is an abnormal expression in nearly 20 different kinds of malignant tumors, and is involved in various biological events such as tumorigenesis, proliferation, differentiation, invasion and metastasis, apoptosis resistance and metabolism through different forms, and by intervention and knockout, It is possible to reverse the malignant phenotype of the tumor after the suppression of the abnormal expression of the CD147 by blocking or the like[2]. With the development of the study, the crystal structure of the CD147 molecule is successfully resolved, and the specific drug screening based on the molecular structure can be made possible. CD147 can be said to have the drug target characteristic of the tumor, yet there is no small molecular medicine to target CD147 at present. To sum up, the purpose of this study is to design and screen anti-tumor micromolecule inhibitors which can target CD147 based on the molecular structure of CD147, and to probe into the molecular mechanism of CD147. This topic mainly includes three parts: the first part, the virtual screening of small molecular compound targeting CD147 and the determination of the pilot compound AC-73. We applied the computer-aided drug screening platform to establish the model of the pharmacophore model based on the three-dimensional crystal structure of CD147. and then performing virtual screening and scoring on the nearly 300,000 small molecular compound from the small molecular compound library of the SPECS, and finally obtaining the 100 candidate small molecule compounds in the comprehensive ranking. A pilot compound AC-73 was further determined by the SPR test and the gelatin enzyme spectrum test for 100 compounds. Finally, when the combination of AC-73 and CD147 is predicted by SPR test, molecular docking simulation technique and point mutation test, the possible binding site is the N-terminal GLUT64 and the GLU73 amino acid residue of the extracellular section of CD147. The second part, small molecule compound AC-73, represses the invasion and metastasis of the liver cancer and the molecular mechanism. In this part of the study, we used CD147 to report more hepatocellular carcinoma as the study model and to explore the function and molecular mechanism of AC-73. Using cell scratch test and invasive cell test, it was found that AC-73 was able to inhibit the migration and invasion of liver cancer cells in vitro, and the reduction of the movement and invasion ability was not caused by AC-73. Further, by comparing the differences in the drug activity of the AC-73 in the wild-type liver cancer cell line and the liver cancer cell line that specifically knocked out the CD147, it is confirmed that the ability of the AC-73 to inhibit the movement and metastasis of the liver cancer cells is achieved by the specific targeting of the CD147. The molecular mechanism of AC-73 was studied by means of Native SDS-PAGE, Western blot, Gelatinase, RT-q PCR and Western blot. The results showed that AC-73 could downregulate the signal pathway of CD147/ ERK1/ 2/ STAT3/ MMP-2 by inhibiting the formation of the CD147 homodimer, thereby inhibiting the invasion and metastasis of the liver cancer cells. The above-mentioned functional characteristics and molecular signal mechanism of AC-73 were verified in nude mouse liver in situ tumor model and confirmed. The in-vivo toxicity test shows that AC-73 is well tolerated in mice, has less toxic and side effect, and has a good biological potential. In the third part, the tumor-inhibiting characteristics of HA-08 and the mechanism of pro-apoptotic mechanism of the modified compound HA-08 were studied. In this part of the exploratory work, we carried out a structural transformation of AC-73 and initially obtained eight modified compounds. A small molecular compound HA-08 capable of interacting with the CD147 was obtained through the molecular docking simulation and the initial screen of the SPR test. The small molecular compound was synthesized and purified for later functional research. The invasion cell test demonstrated that HA-08 could inhibit the invasion and metastasis of the liver cancer cells, and also found that HA-08 has an effect on the activity of the liver cancer cells. Native SDS-PAGE/ Western blot proved that HA-08 could not inhibit CD147 formation of homolog, suggesting that the molecular mechanism of HA-08 was different from that of AC-73. Subsequently, we used WST-1 to test the effect of HA-08 on the activity of 9 different tumor cells, and found that HA-08 has a more common tumor-inhibiting activity on tumor cells. It was found that HA-08 could promote the apoptosis of tumor cells by Annexin V-FITC/ PI flow detection, JC-1 and Hoechst staining, and Western blot. The tumor-inhibiting characteristics and the pro-apoptotic ability of HA-08 were confirmed in the model of tumor-bearing nude mice. Finally, by comparing the sensitivity of HA-08 to tumor cells with different levels of CD147 expression, it is clear that the tumor-inhibiting activity of HA-08 and the ability to promote the apoptosis of tumor cells have a significant correlation with CD147.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5
【参考文献】
相关期刊论文 前1条
1 张弛;满大鹏;马术明;曹树文;李东文;;CD147、OPN和MMP-2在口腔鳞状细胞癌中的表达及意义[J];四川大学学报(医学版);2012年05期
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