基于分子对接和体外大鼠肝微粒体抑制实验综合考察何首乌中潜在肝毒性成分研究
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摘要
目的基于UDP-葡萄糖醛酸转移酶1A1(UGT1A1)介导的胆红素代谢,构建UGT1A1酶蛋白模型,用于研究何首乌中潜在致肝毒性成分的毒性作用,并用体外大鼠肝微粒体抑制实验进行验证。方法采用同源模建方法构建UGT1A1酶蛋白结构,将UGT1A1底物胆红素及何首乌中主要蒽醌类成分大黄素、大黄酚、大黄酸、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄素甲醚与UGT1A1蛋白进行分子对接,考察分子结合靶点及结合强弱。采用大鼠肝微粒体孵育体系(RLM),加入系列浓度的底物胆红素对照品溶液及待测单体对照品溶液,检测表观抑制常数(Ki),测定蒽醌类单体成分对UGT1A1酶的抑制作用。结果分子对接结果显示,UGT1A1酶蛋白结构上共有9个活性口袋区,胆红素的结合口袋确定为位点F;6个单体主要集中在两个活性口袋区:大黄素、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄酚对接进入位点F;大黄素甲醚和大黄酸对接进入位点C。结合于UGT1A1酶蛋白位点C中的单体大黄酸和大黄素甲醚的结合自由能(IE)值较小;位点F区中,单体大黄素-8-O-β-D-葡萄糖苷、大黄素及羟基大黄素具有较高的IE值,结合能力强。体外抑制实验显示,大黄素-8-O-β-D-葡萄糖苷、大黄素表现为较强的竞争型抑制作用,羟基大黄素为较强的混合型抑制作用,与分子对接结果一致。结论大黄素-8-O-β-D-葡萄糖苷、羟基大黄素、大黄素对UGT1A1酶介导的胆红素代谢产生较强的抑制作用,构建的UGT1A1酶蛋白模型可有效预测药物的潜在风险。
Objective Based on UGT1 A1 enzyme-mediated bilirubin metabolism UGT1 A1 enzyme protein was constructed by homology modelling to study the toxic effects of potential hepatotoxic components in Polygonum multiflorum. Methods The UGT1 A1 enzyme protein structure was constructed by homology modeling method. Then bilirubin and the main anthraquinones(emodin, chrysophanol, rhein, hydroxy emodin, emodin-8-O-beta-D-glucoside and emodin methyl ether) in P. multiflorum were molecularly docked with UGT1 A1 protein to investigate the binding target and the action mode. Rat liver microsome incubation system(RLM) was used to determine the inhibitory effect of anthraquinone monomer components on UGT1 A1 enzyme by adding a series of concentration of bilirubin reference solution and monomer reference solution to calculate the apparent inhibitory constant(Ki). Results Molecular docking results showed that there were nine active pocket regions in UGT1 A1 protein structure, and the binding pocket of bilirubin was identified as site F; Six monomers were mainly concentrated in two active pocket regions: emodin,hydroxy emodin, emodin-8-O-beta-D-glucoside and chrysophanol docking entry site F; emodin methyl ether and emodin acid docking entry site C. The binding free energy(IE) of emodin methyl ether and emodin acid in site C of UGT1 A1 enzyme protein was lower. In site F, emodin-8-O-beta-D-glucoside, emodin and hydroxy-emodin had higher IE values and stronger binding ability.in vitro inhibition experiments showed that emodin-8-O-beta-D-glucoside, emodin and hydroxy emodin showed strong competitive inhibition, which was consistent with the results of molecular docking. Conclusion Emodin-8-O-beta-D-glucoside, hydroxy emodin and emodin have strong inhibitory effects on UGT1 A1-mediated bilirubin metabolism. The UGT1 A1 enzyme protein model can effectively predict the potential risks of drugs.
Objective Based on UGT1 A1 enzyme-mediated bilirubin metabolism UGT1 A1 enzyme protein was constructed by homology modelling to study the toxic effects of potential hepatotoxic components in Polygonum multiflorum. Methods The UGT1 A1 enzyme protein structure was constructed by homology modeling method. Then bilirubin and the main anthraquinones(emodin, chrysophanol, rhein, hydroxy emodin, emodin-8-O-beta-D-glucoside and emodin methyl ether) in P. multiflorum were molecularly docked with UGT1 A1 protein to investigate the binding target and the action mode. Rat liver microsome incubation system(RLM) was used to determine the inhibitory effect of anthraquinone monomer components on UGT1 A1 enzyme by adding a series of concentration of bilirubin reference solution and monomer reference solution to calculate the apparent inhibitory constant(Ki). Results Molecular docking results showed that there were nine active pocket regions in UGT1 A1 protein structure, and the binding pocket of bilirubin was identified as site F; Six monomers were mainly concentrated in two active pocket regions: emodin,hydroxy emodin, emodin-8-O-beta-D-glucoside and chrysophanol docking entry site F; emodin methyl ether and emodin acid docking entry site C. The binding free energy(IE) of emodin methyl ether and emodin acid in site C of UGT1 A1 enzyme protein was lower. In site F, emodin-8-O-beta-D-glucoside, emodin and hydroxy-emodin had higher IE values and stronger binding ability.in vitro inhibition experiments showed that emodin-8-O-beta-D-glucoside, emodin and hydroxy emodin showed strong competitive inhibition, which was consistent with the results of molecular docking. Conclusion Emodin-8-O-beta-D-glucoside, hydroxy emodin and emodin have strong inhibitory effects on UGT1 A1-mediated bilirubin metabolism. The UGT1 A1 enzyme protein model can effectively predict the potential risks of drugs.
引文
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[13] Li Y, Jiang L D, Chen X, et al. Discovery of potential ATP-sensitive potassium channel openers with potential hypotensive activity from Chinese herbs based on molecular simulation[J]. China J Chin Mater Med, 2016,41(2):264-271.
[14] Song J H, Cui L, An L B, et al. Inhibition of UDPglucuronosyltransferases(UGTs)activity by constituents of Schisandra chinensis[J]. Phytother Res, 2015, 29(10):1658-1664.
[15] Li L, Huang X J, Peng J L, et al. Wedelolactone metabolism in rats through regioselective glucuronidation catalyzed by uridine diphosphate-glucuronosyl transferases1As(UGT1As)[J]. Phytomedicine, 2016; 23(4):340-349.
[16]汪祺,张玉杰,戴忠,等.微粒体体系中胆红素及其代谢产物测定方法的建立[J].药物分析杂志, 2015, 35(9):1544-1550.
[17]汪祺,戴忠,王亚丹,等.何首乌中8种成分在大鼠肝微粒体体系中的肝毒性研究[J].中国药学杂志, 2018, 53(8):589-593.
[18] Scriver C R. The metabolic&molecular bases of inherited disease[M]. New York:McGraw-Hill, 2001.
[19]焦凌霞,徐茜茜,刘媛,等.基于同源建模与分子对接技术构建抗Bt Cry1类毒素单链抗体定点饱和突变库[J].现代食品科技, 2016, 32(3):12-17.
[20]胡祖权,李和平,张静柏,等.伏马菌素B_1特异单链抗体的同源建模及分子对接模拟研究[J].免疫学杂志,2015, 31(7):618-622.
[21]王明华,李杜娟, Wang Minghua,等.分子对接比较随机RNA片段与同源建模蛋白及其模板的相互作用[J].计算机与应用化学, 2017, 34(7):497-502.
[22]孙彬,刘敏,赵世振,等.白念珠菌几丁质合成酶三维结构的同源建模及其与FR-900403的分子对接研究[J].中国真菌学杂志, 2017, 12(4):193-197, 215.
[23]李军.纤维素酶E4的同源建模和分子对接研究[D].广州:华南理工大学, 2012.
[24]庄绪静.同源建模和分子对接方法的应用与发展[A].中国植物保护学会.植保科技创新\与病虫防控专业化——中国植物保护学会2011年学术年会论文集[C].中国植物保护学会, 2011:7.
[25]王晓欢.基于分子对接的蒽醌类衍生物与c-Abl结合的比较研究[A].天津市生物医学工程学会.天津市生物医学工程学会2007年学术年会论文摘要集[C].天津市生物医学工程学会, 2007:1.
[26]李聃,盛莉,李燕.药物转运体的研究方法[J].药学学报, 2014, 49(7):963-970.
[27] Ritter J K, Chen F, Sheen Y Y, et al. A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDPglucuronosyltransferase isozymes with identical carboxyl termini[J]. J Biol Chem, 1992, 267(5):3257-3261.
[28] Ouzzine M, Magdalou J, Burchell B, et al. An internal signal sequence mediates the targeting and retention of the human UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum[J]. J Biol Chem, 1999, 274(44):31401-31409.
[29] Ramírez J, Mirkov S, House L K, et al. Glucuronidation of OTS167 in humans is catalyzed by UDPglucuronosyltransferases UGT1A1, UGT1A3, UGT1A8,andUGT1A10[J].DrugMetabDispos,2015,43(7):928-935.
[2] Vítek L, Ostrow J D. Bilirubin chemistry and metabolism; harmful and protective aspects[J]. Curr Pharm Des, 2009, 15(25):2869-2883.
[3] Hoekstra L T, de Graaf W, Nibourg G A, et al.Physiological and biochemical basis of clinical liver function tests:A review[J]. Ann Surg, 2013, 257(1):27-36.
[4]刘芳,李波.药物引起肝脏损伤的评价与检测[J].药物分析杂志, 2010, 30(10):1981-1984.
[5] Vítek L, Ostrow J D. Bilirubin chemistry and metabolism; harmful and protective aspects[J]. Curr Pharm Des, 2009, 15(25):2869-2883.
[6] Hinshaw L B. Sepsis/septic shock:participation of the microcirculation:an abbreviated review[J]. Crit Care Med, 1996, 24(6):1072-1078.
[7] Panis B, Wong D R, Hooymans P M, et al. Recurrent toxic hepatitis in a Caucasian girl related to the use of Shou-Wu-Pian, a Chinese herbal preparation[J]. J Pediatr Gastroenterol Nutr, 2005, 41(2):256-258.
[8] Jung K A, Min H J, Yoo S S, et al. Drug-induced liver injury:twenty five cases of acute hepatitis following ingestion of polygonum multiflorum thunb[J]. Gut Liver,2011, 5(4):493-499.
[9]杨倩,李晓宇,赵新妹,等.含何首乌的中成药不良反应系统分析[J].中草药, 2017, 48(9):1878-1887.
[10]鄢良春,赵军宁,邱雄.何首乌安全性问题研究进展[J].中药药理与临床, 2009, 25(3):77-81.
[11] Qiao L S, Jiang L D, Luo G G, et al. Prediction of ETA oligopeptides antagonists from Glycine max based on in silico proteolysis[J]. China J Chin Mater Med, 2017, 42(4):746-751.
[12]张青青,姚其正,张生平,等.靛玉红类CDK1抑制剂的同源模建、分子对接及3D-QSAR研究[J].物理化学学报, 2014, 30(2):371-381.
[13] Li Y, Jiang L D, Chen X, et al. Discovery of potential ATP-sensitive potassium channel openers with potential hypotensive activity from Chinese herbs based on molecular simulation[J]. China J Chin Mater Med, 2016,41(2):264-271.
[14] Song J H, Cui L, An L B, et al. Inhibition of UDPglucuronosyltransferases(UGTs)activity by constituents of Schisandra chinensis[J]. Phytother Res, 2015, 29(10):1658-1664.
[15] Li L, Huang X J, Peng J L, et al. Wedelolactone metabolism in rats through regioselective glucuronidation catalyzed by uridine diphosphate-glucuronosyl transferases1As(UGT1As)[J]. Phytomedicine, 2016; 23(4):340-349.
[16]汪祺,张玉杰,戴忠,等.微粒体体系中胆红素及其代谢产物测定方法的建立[J].药物分析杂志, 2015, 35(9):1544-1550.
[17]汪祺,戴忠,王亚丹,等.何首乌中8种成分在大鼠肝微粒体体系中的肝毒性研究[J].中国药学杂志, 2018, 53(8):589-593.
[18] Scriver C R. The metabolic&molecular bases of inherited disease[M]. New York:McGraw-Hill, 2001.
[19]焦凌霞,徐茜茜,刘媛,等.基于同源建模与分子对接技术构建抗Bt Cry1类毒素单链抗体定点饱和突变库[J].现代食品科技, 2016, 32(3):12-17.
[20]胡祖权,李和平,张静柏,等.伏马菌素B_1特异单链抗体的同源建模及分子对接模拟研究[J].免疫学杂志,2015, 31(7):618-622.
[21]王明华,李杜娟, Wang Minghua,等.分子对接比较随机RNA片段与同源建模蛋白及其模板的相互作用[J].计算机与应用化学, 2017, 34(7):497-502.
[22]孙彬,刘敏,赵世振,等.白念珠菌几丁质合成酶三维结构的同源建模及其与FR-900403的分子对接研究[J].中国真菌学杂志, 2017, 12(4):193-197, 215.
[23]李军.纤维素酶E4的同源建模和分子对接研究[D].广州:华南理工大学, 2012.
[24]庄绪静.同源建模和分子对接方法的应用与发展[A].中国植物保护学会.植保科技创新\与病虫防控专业化——中国植物保护学会2011年学术年会论文集[C].中国植物保护学会, 2011:7.
[25]王晓欢.基于分子对接的蒽醌类衍生物与c-Abl结合的比较研究[A].天津市生物医学工程学会.天津市生物医学工程学会2007年学术年会论文摘要集[C].天津市生物医学工程学会, 2007:1.
[26]李聃,盛莉,李燕.药物转运体的研究方法[J].药学学报, 2014, 49(7):963-970.
[27] Ritter J K, Chen F, Sheen Y Y, et al. A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDPglucuronosyltransferase isozymes with identical carboxyl termini[J]. J Biol Chem, 1992, 267(5):3257-3261.
[28] Ouzzine M, Magdalou J, Burchell B, et al. An internal signal sequence mediates the targeting and retention of the human UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum[J]. J Biol Chem, 1999, 274(44):31401-31409.
[29] Ramírez J, Mirkov S, House L K, et al. Glucuronidation of OTS167 in humans is catalyzed by UDPglucuronosyltransferases UGT1A1, UGT1A3, UGT1A8,andUGT1A10[J].DrugMetabDispos,2015,43(7):928-935.