酶生物色谱法在氨肽酶N抑制剂筛选中的应用研究
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摘要
氨肽酶N(APN)与原发肿瘤和继发肿瘤的生长、血管生成密切相关,甚至对肿瘤细胞的增殖分化过程亦起促进作用,抑制APN的活性可以有效的阻断肿瘤的侵袭和转移,因此,APN已成为抗肿瘤药物研究的靶点之一。酶生物色谱法是分子生物色谱法的一种,它以酶为色谱固定相,使效应成分的分离与筛选结合在一起,可以从混合物中筛选特定的效应成分。本课题以APN为靶点,制备了新的APN固定相,建立了APN的酶生物色谱法,为抗肿瘤药物的研究开发提供了新的筛选模型。全文分四章,主要研究工作和成果如下：
1.采用组织提取法,以猪肾为原料,制备APN,采用盐析、柱层析技术进行了纯化,并对其生物化学和有关酶学性质进行了研究。结果表明,酶经纯化后比活为10.08 U/mg,酶得到了较好的分离纯化,能满足实验的要求。APN最适温度为50℃,在50～55℃范围内相对稳定性；最适pH为7.8,偏弱碱性；阳性抑制剂Bestatin的半数有效抑制浓度为10.351μM；以L-亮氨酰-p-硝基苯胺为底物,测得其Km值为0.55 mM。
2.制备硅胶整体柱,并进行包覆和修饰,以其为载体对APN的固定化条件进行了研究,并对固定化APN的性质进行了研究。结果表明酶固定化的最佳pH为7.8,酶量为40 ml,反应时间为6 h；酶经固定化后,最适pH不变,仍为7.8,pH稳定范围增加,最适温度为55℃,稳定性增加。
3.建立APN的酶生物色谱筛选模型,以Bestatin为阳性对照,对模型进行了验证,并采用此模型对35个新化合物进行了筛选。结果表明,在该筛选模型中,Bestatin对APN的抑制作用具有明显的量-效关系,存在浓度依赖性,证明该筛选模型可行；采用此模型进行筛选,为进一步的化合物结构修饰提供有效信息。
Aminopeptidase N (APN) is closely related to the growth of primary and secondary tumors and the creation of new blood vessel, even is contributed to the proliferation and differentiation of tumor cells. The invasion and metastasis of tumors can be effectively blocked by the inhibition of APN activity. APN has become to be an attractive target of research on anticancer. Enzyme biochromatography is a biochromatography, which takes enzymes for chromatographic stationary phase, integrates the separation and screening of active components and can directly screen specific components from mixtures. This paper studied on the preparation of new APN stationary phase and the establishment of APN biochromatography with APN as target, providing a new screening model for anticancer drug research. This paper contains four chapters, The main research work and achievements are summarized as follows:
1. APN was prepared by tissue extraction with porcine kidney as raw materials and purified by salt out and column chromatography. Then the biological chemistry and related properties of APN were studied. The results showed that APN was well separated and purified and the specific activity was 10.08 U/mg, which can satisfy the requirement of experiment. The optimal temperature of APN was 50℃, and it was relatively stable between 50～55℃. The optimal pH was 7.8, which was weakly alkaline. The IC50 of APN inhibitor Bestatin was 10.35μM. The Km of APN was 0.55 mM, with L-leucine-p-nitroanilide as substrate.
2. Monolithic silica columns were prepared, clad and modified before used as the carrier. The conditions of the immobilization of APN and the properties of immobilized APN were studied. The results showed that the optimum conditions to immobilize APN were:pH 7.8, the quantity of enzyme was 40 ml and the reaction time was 6 h. After immobilization, the optimal pH of immobilized APN was also7.8, the stability limits of pH was expanded. The optimal temperature of immobilized APN was 55℃. The stability of immobilized APN was increased.
3. The biochromatography screening model of APN was established, and was validated by Bestatin.35 new compounds were screened by this screening model. The results showed that in this screening model, dose-response relationship of Bestatin to APN activity was observed, the inhibition rate of Bestatin to APN activity was depend on the concentration of Bestatin, proved the screening model was feasible. The screening results provided effective information for further compounds structure modification.
1.采用组织提取法,以猪肾为原料,制备APN,采用盐析、柱层析技术进行了纯化,并对其生物化学和有关酶学性质进行了研究。结果表明,酶经纯化后比活为10.08 U/mg,酶得到了较好的分离纯化,能满足实验的要求。APN最适温度为50℃,在50～55℃范围内相对稳定性；最适pH为7.8,偏弱碱性；阳性抑制剂Bestatin的半数有效抑制浓度为10.351μM；以L-亮氨酰-p-硝基苯胺为底物,测得其Km值为0.55 mM。
2.制备硅胶整体柱,并进行包覆和修饰,以其为载体对APN的固定化条件进行了研究,并对固定化APN的性质进行了研究。结果表明酶固定化的最佳pH为7.8,酶量为40 ml,反应时间为6 h；酶经固定化后,最适pH不变,仍为7.8,pH稳定范围增加,最适温度为55℃,稳定性增加。
3.建立APN的酶生物色谱筛选模型,以Bestatin为阳性对照,对模型进行了验证,并采用此模型对35个新化合物进行了筛选。结果表明,在该筛选模型中,Bestatin对APN的抑制作用具有明显的量-效关系,存在浓度依赖性,证明该筛选模型可行；采用此模型进行筛选,为进一步的化合物结构修饰提供有效信息。
Aminopeptidase N (APN) is closely related to the growth of primary and secondary tumors and the creation of new blood vessel, even is contributed to the proliferation and differentiation of tumor cells. The invasion and metastasis of tumors can be effectively blocked by the inhibition of APN activity. APN has become to be an attractive target of research on anticancer. Enzyme biochromatography is a biochromatography, which takes enzymes for chromatographic stationary phase, integrates the separation and screening of active components and can directly screen specific components from mixtures. This paper studied on the preparation of new APN stationary phase and the establishment of APN biochromatography with APN as target, providing a new screening model for anticancer drug research. This paper contains four chapters, The main research work and achievements are summarized as follows:
1. APN was prepared by tissue extraction with porcine kidney as raw materials and purified by salt out and column chromatography. Then the biological chemistry and related properties of APN were studied. The results showed that APN was well separated and purified and the specific activity was 10.08 U/mg, which can satisfy the requirement of experiment. The optimal temperature of APN was 50℃, and it was relatively stable between 50～55℃. The optimal pH was 7.8, which was weakly alkaline. The IC50 of APN inhibitor Bestatin was 10.35μM. The Km of APN was 0.55 mM, with L-leucine-p-nitroanilide as substrate.
2. Monolithic silica columns were prepared, clad and modified before used as the carrier. The conditions of the immobilization of APN and the properties of immobilized APN were studied. The results showed that the optimum conditions to immobilize APN were:pH 7.8, the quantity of enzyme was 40 ml and the reaction time was 6 h. After immobilization, the optimal pH of immobilized APN was also7.8, the stability limits of pH was expanded. The optimal temperature of immobilized APN was 55℃. The stability of immobilized APN was increased.
3. The biochromatography screening model of APN was established, and was validated by Bestatin.35 new compounds were screened by this screening model. The results showed that in this screening model, dose-response relationship of Bestatin to APN activity was observed, the inhibition rate of Bestatin to APN activity was depend on the concentration of Bestatin, proved the screening model was feasible. The screening results provided effective information for further compounds structure modification.
引文
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[2]. I.Ssiki, H.fujii, J.Yoneda, et al. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation. Int. J. Cancer.1993;54:137-143.
[3]. S.V. Bhagwat, J.Lahdenranta, R. Giordano,et al. CD13/APN is activated by angiogenic signals and is essential for capillary tube formation. Blood.2001; 97:652-659.
[4]. R. Pasqualini, E. Koivunen, R. Kain, et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res.2000; 60:722-727'.
[5]. C. Antczak, M. I. De, B. Bauvois. Ectopeptidases in pathophysiology. Bioessays 2001,23,(3): 251-260.
[6]. N. Luciani, C. Marie-Claire, E. Ruffet, et al. Characterization of Glu350 as a Critical Residue Involved in the N-Terminal Amine Binding Site of Aminopeptidase N (EC 3.4.11.2):Insights into Its Mechanism of Action. Biochemistry 1998,37 (2):686-692.
[7]. J. Grembecka, A. Mucha, T. Cierpicki, P. Kafarski. The Most Potent Organophosphorus Inhibitors of Leucine Aminopeptidase. Structure-Based Design, Chemistry, and Activity. J. Medicinal Chemistry 2003,46 (13):2641-2655.
[8]. N. M. Hooper. Families of zinc metalloproteases. FEBS Lett.1994,354 (1):1-6.
[9]. B. Nocek, R. Mulligan, M. Bargassa, et al. Crystal structure of aminopeptidaseN from human pathogenNeisseria meningitidis. Proteins:Structure, Function, and Bioinformatics. 2007,70 (1):273-279.
[10]. P.C. Rudberg, F. Tholander, M. Thunnissen,et al. Leukotriene A(4) hydrolase/aminopeptidase-Glutamate 271 is a catalytic residue with specific roles in two distinct enzyme mechanisms. Journal of Biological Chemistry.2002,277(2):1398-1404.
[11]. W. Jiang, J. S. Bond. Families of metalloendopeptidases and their relationships. FEBS Lett.1992, 312(2):100-114.
[12].D.G. Hangauer, A.F. Monzingo, B.W. Matthews. An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides. Biochemistry. 1984,23(24):5730-5741.
[13]. K. Ito, Y. Nakajima, Y. Onohara, et al. Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition. J Biol Chem.2006,281(44):33664-33676.
[14]. L.A. Liotta, P.S. Steeg. Cancer metastasis and angiogenesis:an imbalance of positive and negative regulation. Cell.1991,64 (2):327-336.
[15]. Y. Kitamura, M. Watanabe, S. Komatsubara,et al. Urinary excretion of glycine prolile dipeptidile aminopeptidase, N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase and low molecular protein in patients with renal cell carcinoma. Hinyokika Kiyo.1990,36(5):535-539.
[16]. H. Hashida, A. Takabayashi, M. Kanai, et al. Aminopeptidase N is involved in cell motility and angiogenesis:Its clinical significance in human colon cancer. Gastroenterology.2002,122(2):376-386.
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