-
细胞色素P450(cytochrome,CYP450)是一种需要亚铁血红素(HEM)活化氧,以硫醇盐结合血红素为活性中心催化氧原子转移的氧化还原酶[1-2]。活性中心HEM的铁离子一侧与半胱氨酸的硫络合,另一侧可与水中的氧分子络合。半腕氨酸可与亚铁血红素中的铁元素形成硫醇盐离子键,成为铁的一个配体。这也是细胞色素P450中的铁能与一氧化碳络合,并在450 nm存在特征吸收的原因。
CYP3A4是CYP家族中最重要的一种,其活性在人类肝脏中占总CYP活性的60%,CYP3A4酶含量及酶活性存在较大的个体差异。CYP3A4在肝脏表达量最丰富,在人前列腺、乳腺、消化道、结肠及小肠组织液同样有很高的表达。现已经发现CYP3A4参与多种药物的代谢,并且在人群中广泛存在着遗传多态性,是引起个体间和种族间对同一底物代谢能力不同的原因之一。
己烯雌酚(Diethylstilbestrol,DES)作为一种合成的雌激素,具有比雌二醇更强的雌激素活性[3-4]。20世纪40年代起,己烯雌酚主要作为鸡、牛等的增肉剂,鱼类的生长促进剂[5-6],它在生物体内不易降解,会通过食物链富集,严重危害到人类健康[7]。在上世纪七八十年代,己烯雌酚作为类雌激素药物广泛运用于口服避孕,以及防止流产。后有研究发现其是一种内分泌干扰物,DES暴露对人类和动物有很强的负面影响。1971年,己烯雌酚被发现可诱发多种女性疾病遭到禁用[8]。1998年,蒋义国等发现己烯雌酚可能是一种促癌因子,会提高其他致癌物质的致癌性[9]。有研究发现孕期服用DES会影响胎儿生殖器官发育,也可能增加胎儿患生殖道癌症的风险。
DES是脂溶性物质,容易在动物体内残留,即使排出体外也会在水源和土壤中富集,造成环境污染恶性循环,并且食用含有DES残留的动物产品也会增加患癌的风险[10]。Hu等通过计算模拟研究己烯雌酚及其类似物对胰腺脂肪酶作用机理,结果得到己烯雌酚及其类似物对胰腺脂肪酶有抑制作用,可能对哺乳动物脂质吸收和体重增加起重要作用[11]。
由于DES的药理作用及其成本较低,DES仍然用于临床实践和动物中,CYP3A4酶作为一种重要的药物代谢酶,参与多种药物代谢。本文采用计算模拟和光谱法,从理论和实验来探究在模拟人体环境条件下,DES和CYP3A4酶的相互作用机制。从分子水平了解CYP3A4和DES的相互作用,为进一步研究DES对生命体造成的危害及其毒理作用打下基础。
结合光谱法和计算模拟分析CYP3A4酶与己烯雌酚的作用机制
Interaction between Diethylstilbestrol and CYP3A4 based on multi-spectrum methods and computational simulations
-
摘要: 本文结合分子对接、动力学模拟和光谱法,研究了己烯雌酚(Diethylstilbestrol,DES)与CYP3A4酶在模拟生理环境下(pH7.4)的相互作用机制。利用分子对接和动力学模拟方法从理论上模拟CYP3A4与DES相互作用后的构象变化情况。通过光谱法从实验角度进行验证,荧光光谱得出DES对CYP3A4的猝灭机制属于静态猝灭,红外和紫外光谱结果表明,DES与CYP3A4结合后,改变了CYP3A4的内部环境和构象。理论模拟和实验结果相互印证,为探究DES和CYP3A4的相互作用提供了重要信息和参考依据。Abstract: The interaction mechanism between Diethylstilbestrol (DES) and CYP3A4 enzyme in simulated physiological environment (pH=7.4) was studied by molecular docking, kinetic simulation and spectrometric method. The molecular docking and dynamics simulation were used to simulate the conformation changes of CYP3A4 and DES in theory. Then, it was experimental verified by spectral methods. The fluorescence spectra showed that the quenching mechanism of CYP3A4 by DES was static quenching. The results of IR and UV spectra showed that the internal environment and conformation of CYP3A4 were changed after DES interacted with CYP3A4. The theoretical simulation and experimental results were consistent with each other, which provides important information and reference for exploring the interaction between DES and CYP3A4.
-
Key words:
- CYP3A4 /
- diethylstilbestrol /
- multi-spectrum methods /
- computational simulation
-
图 5 (a)含有不同浓度DES的CYP3A4体系的UV吸收光谱;(b)在λ190—450 nm范围内的紫外吸收光谱。实线表示CYP3A4酶(Ⅰ,黑色)、DES(Ⅱ,红色)和CYP3A4-DES复合物(1∶1)(Ⅲ,蓝色)的UV光谱。虚线表示每个光谱的理论吸光度。
Figure 5. (a) UV absorption spectra of CYP3A4 system with different concentrations of DES (b) UV absorption spectra in range of λ190—450 nm. The solid lines represent the UV spectra of the CYP3A4 (Ⅰ,black), DES (Ⅱ,red) and the CYP3A4-DES complex (1∶1) (Ⅲ,blue). The dotted lines represent the theoretical absorbance of each spectra.
表 1 CYP3A4与DES氢键作用力
Table 1. Hyrrogen Bond between CYP3A4 and DES
氨基酸残基Amino acid residues 作用原子Atom 键长/nm Bond length ILE301 O—O 0.259 ALA305 N—O 0.302 PHE108 O—O 0.232 表 2 CYP3A4和DES在不同温度下相互作用的猝灭常数(Ksv),生物猝灭速率常数(kq)
Table 2. Quenching constant and biomolecule quenching rate constant of the interaction between CYP3A4 and DES at different temperatures.
温度Temperature/K Ksv(×108 )/(L·mol−1) kq(×1018)/( L·mol−1·s−1) 290 8.77 8.77 300 4.05 4.05 310 2.11 2.11 -
[1] SHAIK S, FILATOV M, SCHRDER D, et al. Electronic structure makes a difference: Cytochrome P-450 mediated hydroxylations of hydrocarbons as a two-state reactivity paradigm [J]. Chemistry A European Journal, 2010, 4(2): 193-199. [2] MEUNIER B, VISSER S P D, SHAIK S. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzyme [J]. Cheminform, 2004, 104(9): 3947-3980. [3] KOONG L Y, WASTON C S. Direct estradiol and diethylstilbestrol actions on early-versus late-stage prostate cancer cells [J]. The Prostate, 2014, 74(16): 1589-1603. doi: 10.1002/pros.22875 [4] GABIZON, ALBERTO. Diethylstilbestrol for the treatment of patients with castration-resistant prostate cancer: Retrospective analysis of a single institution experience [J]. Oncology Reports, 2014, 31(1): 428-34. doi: 10.3892/or.2013.2852 [5] STEFANICK M L. Estrogens and progestins: Background and history, trends in use, and guidelines and regimens approved by the US Food and drug Administration [J]. American Journal of Medicine, 2005, 118(12): 64-73. doi: 10.1016/j.amjmed.2005.09.059 [6] YIN Y, LIN C X, MA L. Msx2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol [J]. Molecular Endocrinology, 2006, 20(7): 1535-1546. doi: 10.1210/me.2005-0451 [7] 端正花, 朱琳, 宫知远, 等. 己烯雌酚在斑马鱼胚胎中的生物蓄积及毒性机制研究 [J]. 环境科学, 2009, 30(2): 522-526. doi: 10.3321/j.issn:0250-3301.2009.02.036 DUAN Z H, ZHU L, GONG Z Y, et al. Bioaccumulation and toxicity test of diethylstilbestrol to zebrafish (danio rerio) embryo [J]. Environmental Science, 2009, 30(2): 522-526(in Chinese). doi: 10.3321/j.issn:0250-3301.2009.02.036
[8] HE S H, LIANG Z H, ZHAN W, et al. Establishment of the recombinant yeast assay system for environmental estrogens [J]. Journal of Environment & Health, 2002, 19(1): 57-59. [9] 蒋义国, 陈家坤. 己烯雌酚对乌拉坦诱发小鼠肺肿瘤的促进作用 [J]. 癌变. 畸变. 突变, 1998, 10(2): 72-75. JIANG Y G, CHEN J K. Effect of diethylstilbestrol on urethan-induced lung tumorigenesis in kunming mice [J]. Carcinogenesis Teratogenesis and Mutagenesis, 1998, 10(2): 72-75(in Chinese).
[10] 黄芬, 叶绍辉, 龚振明. 己烯雌酚的研究进展 [J]. 中国畜牧兽医, 2007, 34(2): 52-55. HUANG F, YE S H, GONG Z M. The development of diethylstilbestrol [J]. China Animal Husbandry & Veterinary Medicine, 2007, 34(2): 52-55(in Chinese).
[11] HU Q, GUAN X Q, SONG L L, et al. Inhibition of pancreatic lipase by environmental xenoestrogens [J]. Ecotoxicology and Environmental Safety, 2020, 192: 110305. doi: 10.1016/j.ecoenv.2020.110305 [12] 刘红艳, 谢世伟, 唐琳, 等. 基于计算模拟与响应面分析漆酶对己烯雌酚的降解作用 [J]. 环境科学学报, 2020, 40(4): 1174-1184. LIU H Y, XIE S W, TANG L, et al. Degradation of diethylstilbestrol by laccase based on computational simulation and response surface analysis [J]. Acta Scientiae Circumstantiae, 2020, 40(4): 1174-1184(in Chinese).
[13] WEI Y, YI Z S, XU J, et al. Study on the binding characteristics of hydroxylated polybrominated diphenyl ethers and thyroid transporters using the multispectral technique and computational simulation [J]. Journal of Biomolecular Structure & Dynamics, 2018, 37(6): 1402-1413. [14] TANG B, TANG P X, HE J W, et al. Characterization of the binding of a novel antitumor drug ibrutinib with human serum albumin: Insights from spectroscopic, calorimetric and docking studies. [J]. Journal of Photochemistry and Photobiology. B, Biology, 2018, 184: 18-26. doi: 10.1016/j.jphotobiol.2018.05.008 [15] MORRIS G M, HUEY R, LINDSTROM W, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility [J]. Journal of Computational Chemistry, 2010, 30(16): 2785-2791. [16] NASROLLAH R G, AZADEN E H, ALI M M, et al. Effect of polyamines on the structure, thermal stability and 2, 2, 2-trifluoroethanol-induced aggregation of alpha-chymotrypsin. [J]. International Journal of Biological Macromolecules, 2007, 41(5): 597-604. doi: 10.1016/j.ijbiomac.2007.07.018 [17] SADEGH F, BEHZAD S, ALI A S, et al. The influence of putrescine on the structure, enzyme activity and stability of α-chymotrypsin [J]. RSC Advances, 2016, 6(35): 29264-29278. doi: 10.1039/C5RA25053F [18] 董露, 易忠胜, 伍智蔚, 等. 结合光谱法和计算模拟多角度分析2, 2′, 4, 4′, 5-五溴二苯醚与人血清白蛋白的作用机制 [J]. 环境科学学报, 2016, 36(1): 332-339. DONG L, YI Z S, WU Z W, et al. Interaction between 2, 2', 4, 4', 5-pentabromodiphenyl ether and human serum albumin based on multi-spectroscopic and computational simulations [J]. Acta Scientiae Circumstantiae, 2016, 36(1): 332-339(in Chinese).
[19] 李志娟, 韦明奉, 韦勇, 等. 分子荧光光谱法结合分子动力学模拟研究2'-OH-BDE-68与HSA的相互作用 [J]. 理化检验-化学分册, 2017, 53(1): 6-11. LI Z J, WEI M F, WEI Y, et al. Study on the interaction between 2'-OH-BDE-68 and HAS by molecular fluorospectrometry and molecular dynamics simulation [J]. Physical Testing and Chemical Analysis Part B:Chemical Analysis, 2017, 53(1): 6-11(in Chinese).
[20] 孙梅香, 刘文, 高嘉苓, 等. 荧光光谱法研究草甘膦与腐殖酸的相互作用 [J]. 环境化学, 2015, 34(8): 1529-1534. doi: 10.7524/j.issn.0254-6108.2015.08.2014122401 SUN M X, LIU W, GAO J L, et al. Study on the interaction between glyphosate and humic acid using fluorescence spectrometry and parallel factor analysis method [J]. Environmental Chemistry, 2015, 34(8): 1529-1534(in Chinese). doi: 10.7524/j.issn.0254-6108.2015.08.2014122401
[21] 梁彦秋, 邓斌, 刘婷婷, 等. 4-硝基苯胺与人血清白蛋白相互作用的研究 [J]. 环境化学, 2007, 26(6): 845-849. doi: 10.3321/j.issn:0254-6108.2007.06.027 LIANG Y Q, DENG B, LIU T T, et al. Investigation on interaction of 4-nitroaniline with human serum albumin [J]. Environmental Chemistry, 2007, 26(6): 845-849(in Chinese). doi: 10.3321/j.issn:0254-6108.2007.06.027
[22] NAYARA S D A C, CARO P C, DANIEL P B, et al. An investigation into the interaction between piplartine (piperlongumine) and human serum albumin [J]. Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy, 2019, 220: 117084. [23] VERLAG S. Principles of fluorescence spectroscopy [J]. Die Naturwissenschaften, 2006, 78(10): 456. [24] CARUSO I P, FILHO J M B, ARAUJO A S D, et al. An integrated approach with experimental and computational tools outlining the cooperative binding between 2-phenylchromone and human serum albumin [J]. Food Chemistry, 2016, 196: 935-942. doi: 10.1016/j.foodchem.2015.10.027 [25] CARUSO, I P, VILEGAS W, DE S F P, et al. Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays [J]. Journal of Pharmaceutical & Biomedical Analysis, 2014, 98: 100-106. [26] YUE Y Y, ZHAO S F, SUN Y Y, et al. Effects of plant extract aurantio-obtusin on pepsin structure: Spectroscopic characterization and docking simulation [J]. Journal of Luminescence, 2017, 187: 333-339. doi: 10.1016/j.jlumin.2017.03.041 [27] CAMARGO C R, CARUSO I P, GUTIERREZ S J C, et al. Spectral and computational features of the binding between riparins and human serum albumin [J]. Spectrochim Acta A Mol Biomol Spectrosc, 2018, 190: 81-88. doi: 10.1016/j.saa.2017.08.068 [28] MANOUCHEHRI F, IZADMANESH Y, AGHAEE E, et al. Experimental, computational and chemometrics studies of BSA-vitamin B6 interaction by UV–Vis, FT-IR, fluorescence spectroscopy, molecular dynamics simulation and hard-soft modeling methods [J]. Bioorganic Chemistry, 2016, 68(Complete): 124-136. [29] LITVINOV R, FAIZULLIN D, ZUEV Y, et al. The α-helix to β-sheet transition in stretched and compressed hydrated fibrin clots [J]. Biophysical Journal, 2012, 103(5): 1020-1027. doi: 10.1016/j.bpj.2012.07.046 [30] MOZHGAN M, BEHZAD S, ALI A S, et al. Comparative studies on the interaction of spermidine with bovine trypsin by multispectroscopic and docking methods [J]. Journal of Physical Chemistry B Condensed Matter Materials Surfaces Interfaces & Biophysical, 2016, 120(36): 9632-41. [31] SIDDIQI M K, ALAM P, CHATURVEDI S K, et al. Probing the interaction of cephalosporin antibiotic–ceftazidime with human serum albumin: A biophysical investigation [J]. International Journal of Biological Macromolecules, 2017, 105(1): 292-299. [32] ALAM P, CHATURVEDI S K, ANWAR T, et al. Biophysical and molecular docking insight into the interaction of cytosine β-D arabinofuranoside with human serum albumin [J]. Journal of Luminescence, 2015, 164: 123-130. doi: 10.1016/j.jlumin.2015.03.011