靶向CD147的小分子抑制劑的篩選及抗腫瘤機制研究
發(fā)布時間：2019-01-20 08:38
【摘要】：惡性腫瘤是世界范圍內(nèi)主要致死性疾病之一,亦是嚴(yán)重威脅人類生命健康的常見疾病[1]。惡性腫瘤異常增殖、抵抗凋亡以及侵襲轉(zhuǎn)移等生物學(xué)特性,給腫瘤治療帶來了巨大挑戰(zhàn)。傳統(tǒng)的治療手段尚無法從根本上治療惡性腫瘤,且多伴隨明顯毒副作用,給患者帶來痛苦,影響患者生活質(zhì)量。近年來,分子靶向治療作為腫瘤治療的新策略,以其特異性強,副作用小等治療優(yōu)勢逐漸被推崇,許多分子靶向藥物已獲FDA批準(zhǔn)投入臨床,并取得了較好的臨床療效。分子靶向治療策略的核心是靶點的確定,通過對腫瘤分子機制不斷的深入研究,尋找精準(zhǔn)靶點,是攻克腫瘤的關(guān)鍵所在。免疫球蛋白超家族成員CD147分子是近年來報道較多的腫瘤相關(guān)分子(Tumor associated molecule,TAM)。研究表明,CD147分子在近20種不同來源的惡性腫瘤中呈現(xiàn)異常表達(dá),并且通過不同的形式參與腫瘤發(fā)生,增殖,分化,侵襲轉(zhuǎn)移,凋亡抵抗以及代謝等多種生物學(xué)事件,而通過干涉、敲除、阻斷等方式抑制CD147的異常表達(dá)后,可以逆轉(zhuǎn)腫瘤的惡性表型[2]。隨著研究的深入,又成功解析了CD147分子的晶體結(jié)構(gòu),從而使基于分子結(jié)構(gòu)的特異性藥物篩選成為可能�？梢哉fCD147已經(jīng)具備了腫瘤的藥物靶點特性,然而目前尚無靶向CD147的小分子藥物。綜上所述,本課題旨在基于CD147的分子結(jié)構(gòu),設(shè)計篩選出能夠靶向CD147的抗腫瘤小分子抑制劑,并對其分子機制進(jìn)行深入探討。本課題主要包括三部分工作:第一部分,靶向CD147的小分子化合物的虛擬篩選及先導(dǎo)化合物AC-73的確定。我們應(yīng)用計算機輔助藥物篩選平臺,以CD147的三維晶體結(jié)構(gòu)為模型進(jìn)行藥效團(tuán)模型的建立。隨后對來自SPECS小分子化合物庫中的近30萬小分子化合物進(jìn)行虛擬篩選和評分,最終得到綜合排名靠前的100個候選小分子化合物。進(jìn)一步通過SPR試驗以及明膠酶譜試驗對100個化合物進(jìn)行初篩,確定了一個先導(dǎo)化合物AC-73。最后我們通過SPR試驗,分子對接模擬技術(shù)以及點突變等試驗預(yù)測AC-73與CD147結(jié)合時,可能存在的結(jié)合位點為CD147胞外段N端GLU64以及GLU73位氨基酸殘基。第二部分,小分子化合物AC-73阻抑肝癌侵襲轉(zhuǎn)移及分子機制研究。這一部分研究中,我們用CD147參與的惡性腫瘤中報道較多的肝細(xì)胞癌為研究模型,探討AC-73的功能及分子機制。應(yīng)用細(xì)胞劃痕試驗和侵襲小室試驗,發(fā)現(xiàn)AC-73能夠在體外抑制肝癌細(xì)胞遷移運動和侵襲轉(zhuǎn)移,且該運動和侵襲能力的降低并非由AC-73影響了細(xì)胞活性所致。進(jìn)一步通過比較AC-73在野生型肝癌細(xì)胞系和特異性敲除CD147的肝癌細(xì)胞系的藥物活性的差異,證實AC-73抑制肝癌細(xì)胞運動和轉(zhuǎn)移的能力是通過特異性靶向CD147實現(xiàn)的。應(yīng)用Native SDS-PAGE/Western blot、明膠酶譜、RT-q PCR、蛋白免疫印跡等試驗,對AC-73分子機制進(jìn)行研究,結(jié)果表明AC-73可以通過抑制CD147同源二聚體形成的方式,下調(diào)CD147/ERK1/2/STAT3/MMP-2分子信號通路,從而抑制肝癌細(xì)胞發(fā)生侵襲轉(zhuǎn)移。AC-73的上述功能特性和分子信號機制,在裸鼠肝原位移植瘤模型內(nèi)進(jìn)行了驗證,并得到了證實。體內(nèi)毒性試驗顯示,AC-73在小鼠體內(nèi)耐受性較好,毒副作用小,具有良好的成藥潛能。第三部分,AC-73改造化合物HA-08的抑瘤特性及促凋亡機制研究。在這一部分探索性工作中,我們對AC-73進(jìn)行了結(jié)構(gòu)改造,初期獲得了八個改造化合物。經(jīng)過分子對接模擬以及SPR試驗初篩,得到一個能夠與CD147相互作用的小分子化合物HA-08。對該小分子化合物進(jìn)行了合成及純化,用于后期的功能研究。侵襲小室試驗證明,HA-08可以抑制肝癌細(xì)胞發(fā)生侵襲轉(zhuǎn)移,同時發(fā)現(xiàn)HA-08對肝癌細(xì)胞活性產(chǎn)生了影響。Native SDS-PAGE/Western blot試驗證實HA-08不能夠抑制CD147形成同源二聚,提示HA-08的分子機制與AC-73存在差異。隨后,我們應(yīng)用WST-1試驗檢測了HA-08對9種不同腫瘤細(xì)胞活性影響,發(fā)現(xiàn)HA-08對腫瘤細(xì)胞具有較普遍的抑瘤活性。通過Annexin V-FITC/PI流式檢測、JC-1和Hoechst染色以及Western blot等試驗,發(fā)現(xiàn)HA-08能夠促進(jìn)腫瘤細(xì)胞發(fā)生凋亡,主要體現(xiàn)為誘導(dǎo)凋亡表型的出現(xiàn)以及凋亡相關(guān)蛋白水平的變化。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證實。最后我們通過比較HA-08在CD147表達(dá)水平不同的腫瘤細(xì)胞的敏感性差異,明確HA-08的抑瘤活性和促腫瘤細(xì)胞凋亡的能力與CD147呈現(xiàn)顯著相關(guān)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.5
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張弛;滿大鵬;馬術(shù)明;曹樹文;李東文;;CD147、OPN和MMP-2在口腔鱗狀細(xì)胞癌中的表達(dá)及意義[J];四川大學(xué)學(xué)報(醫(yī)學(xué)版);2012年05期
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