本文選題：APN/CD13　切入點：虛擬篩選　出處：《山東大學》2009年博士論文　論文類型：學位論文

【摘要】： 目的:氨肽酶N(Aminopeptidase N,APN),也被稱為CD13,是一類鋅離子依賴性金屬蛋白酶,廣泛存在于小腸、腎及中樞神經(jīng)系統(tǒng)的多種細胞表面。與正常細胞相比,該酶在腫瘤細胞表面高水平表達。研究發(fā)現(xiàn),APN在腫瘤生長、侵襲和轉移過程中發(fā)揮著重要作用。例如,APN可降解細胞外基質,促進原發(fā)腫瘤的生長侵襲,有利于腫瘤的轉移;同時APN還可以促進腫瘤新生血管的形成,是腫瘤新生血管的調節(jié)器;另外該酶還能夠降解胸腺肽和白介素,從而降低機體免疫機能。 正因為APN與腫瘤的密切關系,APN抑制劑的研究已經(jīng)成為抗腫瘤研究領域的一個熱點。Bestatin作為第一個上市的APN抑制劑,臨床上主要用于治療急性成人非淋巴性白血病。近年又發(fā)現(xiàn)許多天然APN抑制劑,如Probestatin,Amastatin,Curcumin等;另外,人們還合成了許多小分子化合物APN抑制劑,如α-氨基磷酸抑制劑,β-氨基硫醇類抑制劑等。本課題組以APN為靶點,經(jīng)十幾年的研究亦報道了許多小分子類肽化合物。 本研究以APN為靶點,在充分調研文獻的基礎上,通過計算機輔助藥物設計技術,設計、合成一系列以L-賴氨酸為基本骨架的小分子化合物,并對它們進行初步的活性篩選,以期發(fā)現(xiàn)具有較好的APN抑制活性的先導化合物。 方法:由APN的晶體結構及抑制劑與酶的作用模式可以看出與APN活性位點相對應,對應的APN抑制劑應由三個部分組成:A疏水性芳環(huán)側鏈;B鋅離子螯合基團位于中間連接片段上;C疏水性芳環(huán)側鏈。A和C兩個疏水側鏈經(jīng)B部分相連。 本研究利用已知的APN抑制劑建立合理的APN受體評價模型,通過常見的鋅離子螯合基團,采用SYBYL/Unity模塊從NCI2000化合物庫中挑選出具有鋅離子螯合基團的化合物。而后將這些化合物與所建立的APN受體評價模型進行對接,挑選出打分較高的化合物作為中靶化合物。在此過程中,庫中原有的APN抑制劑Bestatin(ID:265489)、Phebestin(ID:702307)、para-hydroxybestatin(ID:327461)作為測試分子以驗證方法的有效性。參考中靶化合物和一些已知的APN抑制劑,進而設計了一系列結構全新的以L-賴氨酸為骨架的目標化合物。 本研究將目標化合物進行了體外抑酶實驗、體外腫瘤細胞(HL-60,ES-2,K562,A549,H7402,PLC)生長抑制以及體內抗腫瘤轉移實驗,從中篩選出具有APN抑制活性的抗癌先導化合物。 結果:本文共設計并合成了44個目標化合物,并對所有化合物通過紅外光譜、核磁共振氫譜、電噴霧質譜等方法進行了結構確證。經(jīng)查閱文獻證實,所合成的目標化合物為新型化合物,未見文獻報道。 在C系列化合物中,化合物C7的抑酶活性優(yōu)于兩個陽性對照藥B6和Bestatin,化合物C20的抑酶活性與它們相近。在D系列化合物中,化合物D9、D15的抑酶活性優(yōu)于兩個陽性對照藥B6和Bestatin,化合物D24的抑酶活性與二者相近。 MTT法體外細胞實驗測定了目標化合物對HL-60人白血病細胞株、ES-2卵巢透明癌細胞株、K562人白血病細胞株、A549人肺腺癌細胞株、H7402人肝癌細胞株及PLC人肝癌細胞株的生長抑制作用,結果顯示對APN酶抑制作用較強的化合物C7、D9、D14、D15、D23同樣有很強的HL-60細胞抑制活性,超過了陽性對照藥Bestatin;化合物D14、D21的ES-2細胞抑制活性優(yōu)于陽性對照,D23與之相當;化合物D21的H7402細胞抑制活性優(yōu)于陽性對照,C7、D24與之相當;化合物C7、C20、D9、D14、D15、D19、D21、D23、D24的PLC細胞抑制活性均優(yōu)于陽性對照。 本文在虛擬篩選的基礎上,以所設計、合成的化合物為對象,利用計算機軟件進行了初步的定量構效關系總結。采用比較分子場分析方法(CoMFA)建立了L-賴氨酸類APN抑制劑的定量構效關系(QSAR)模型。通過對CoMFA模型立體場等勢線圖和靜電場等勢線圖的分析,建立的COMFA模型具有較高的交叉驗證系數(shù)q~2和一定的預測能力。 最后,我們還得到一個化合物D24與突變嗜酸熱源菌三角交互作用因子F3的共結晶復合物。 結論:本研究基于APN的晶體結構及抑制劑與酶的作用模式,利用計算機軟件進行設計、合成的L-賴氨酸類化合物具有很好的APN抑制活性。所設計的合成路線科學合理,原料經(jīng)濟易得。通過初步的活性測試發(fā)現(xiàn)了具有進一步研究價值的抗癌先導化合物。其中,化合物C7、D9、D15的抑酶活性優(yōu)于陽性對照藥Bestatin,可作為先導化合物用于指導下一輪的結構優(yōu)化。此外,我們基于化合物結構和活性數(shù)據(jù)建立了有一定預測能力的定量構效關系模型,為今后新型氨肽酶抑制劑的研究奠定了基礎。
[Abstract]:Objective: aminopeptidase N (Aminopeptidase N APN), also known as CD13, is a zinc dependent metalloproteinase, widely exists in the small intestine, the surface of kidney and central nervous system cells. Compared with normal cells, the expression of this enzyme in tumor cells in high level. The study found that APN in tumor growth, plays an important role in the process of invasion and metastasis. For example, APN can degrade extracellular matrix, promoting the growth and invasion of primary tumor, metastasis to tumor; while APN can promote tumor angiogenesis, is a regulator of tumor blood vessels; in addition the enzyme can also degrade thymosin and interleukin, thereby reducing immune function.
Because of the close relationship between APN and tumor, APN inhibitors have become the field of anti-tumor research a hot.Bestatin as the first listed APN inhibitor, is used clinically for the treatment of adult acute non lymphatic leukemia. In recent years, and found that many natural APN inhibitors, such as Probestatin, Amastatin, Curcumin and so on; in addition, people also the synthesis of many small molecules such as APN inhibitors, alpha amino acid inhibitors, beta amino thiol inhibitors. This paper takes APN as the target, after ten years of research also reported a number of small molecular peptide compounds.
This study takes APN as the target, based on a detailed survey of literature, through the design of computer aided drug design, synthesis technology, a series of small molecules with L- lysine as the basic skeleton of the compounds, and they are preliminary screening activity, in order to find lead compounds with better APN inhibitory activity.
Methods: the crystal structure and the mode of action of APN inhibitors and the enzyme can be seen corresponding to the active site of APN, the corresponding APN inhibitors should be composed of three parts: A hydrophobic aromatic side chain; B zinc ion chelating group is located in the middle of the junction fragment; C hydrophobic aromatic side chains of.A and C two hydrophobic side chains are connected via B.
This study uses APN APN receptor inhibitor known to establish reasonable evaluation model, the zinc ion chelating group common, using SYBYL/Unity module selected compounds with zinc ion chelating groups from NCI2000 compound library. And then the APN receptor evaluation of these compounds and the model for docking, pick out as high scoring compounds the target compounds. In this process, the original APN inhibitor Bestatin Library (ID:265489), Phebestin (ID:702307), para-hydroxybestatin (ID:327461) as a test to verify the validity of the molecular method. APN inhibitor compounds and some known reference target, and then designed a series of novel L- lysine as target compounds skeleton.
The aim of this study is to inhibit the enzyme activity in vitro, and inhibit tumor growth in vitro (HL-60, ES-2, K562, A549, H7402, PLC), and in vivo anti-tumor metastasis experiment, and screened out an anticancer lead compound with APN inhibitory activity.
Results: 44 target compounds were designed and synthesized, and all compounds were identified by IR, 1H-NMR and electrospray ionization mass spectrometry.
In the C series of compounds, the enzyme activity of two is better than that of the positive control drug B6 and Bestatin under compound C7, compound C20 inhibitory activity and are similar. The D series compounds, compound D9, enzyme activity of two is better than that of the positive control drug B6 and Bestatin under D15, inhibitory activity of compound D24 is similar two.
The target compounds on human leukemia cell line HL-60 was determined by MTT in vitro, ES-2 ovarian clear cell carcinoma cell line K562, human leukemia cell lines, human lung adenocarcinoma cell line A549, growth inhibition of human hepatocellular carcinoma cell line H7402 and PLC in human hepatocellular carcinoma cell line, showed inhibitory compounds C7, strong effect on APN D14, D15, D9, D23 also has a strong HL-60 cell inhibitory activity than positive control drug Bestatin; compound D14, D21 ES-2 cell inhibitory activity than the positive control, D23 equivalent; compound D21 H7402 cell inhibitory activity than the positive control, C7, D24 and a compound C7; C20, D9, D14, D15, D19, D21, D23, D24, PLC cell inhibitory activity was better than the positive control.
Based on the virtual screening on the design, synthesis of compounds as the object, has carried on the preliminary quantitative structure-activity relationship by using computer software. Using comparative molecular field analysis (CoMFA) to establish a quantitative L- lysine APN inhibitor structure-activity relationship (QSAR) model. Through the analysis of contour maps the CoMFA model of steric contour map and the electrostatic field, the COMFA model has a higher coefficient of cross validation q~2 and predictive ability.
Finally, we also obtained a co crystallization complex of a compound D24 and the trigonometric interaction factor F3 of the mutant eosinophilic bacteria.
Conclusion: This study based on the crystal structure and the mode of action of APN inhibitors and enzymes, were designed using computer software, the synthesis of L- amino acid compound has good inhibitory activity on APN. The synthetic route design is scientific and reasonable, economical and easy to get raw materials. Through the activity of the preliminary test is found to have anticancer lead compounds for further study on value. Among them, compounds C7, D9, better than the activity of the positive control drug Bestatin under D15, can be used as a lead compound for structural optimization under the guidance of a wheel. In addition, we compound structure and activity data based on a quantitative prediction ability of the QSAR model, laid a foundation for further research of new aminopeptidase inhibitors.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R341

【引證文獻】

相關博士學位論文 前1條

1 王學健;小分子類肽APN抑制劑的活性評價及其抗腫瘤作用機制研究[D];山東大學;2010年

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本文編號：1582534