人二氢乳清酸脱氢酶蛋白的表达、纯化及其与新型抑制剂的晶体结构
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摘要
人二氢乳清酸脱氢酶(human dihydroorotate dehydrogenase,hDHODH)是催化嘧啶从头合成途径的一个关键酶。近年来,多种研究表明,抑制该酶可缓解类风湿性关节炎的症状。但该酶的抑制剂甚少,寻找该酶的高效抑制剂具有重要意义。本研究利用PCR技术扩增hDHODH基因,构建重组质粒pET-19b-hDHODH,并在大肠杆菌(Escherichia coli,E.coli) BL21(DE3)中表达,获得可溶性蛋白质。用Ni~(2+)-NTA亲和层析柱对蛋白质进行纯化,获得较高(90%)纯度的hDHODH蛋白,将蛋白质与抑制剂3-(5-乙硫基)-1H-1, 2, 4-三氮唑-3-)苯甲酸和底物DHO混合孵育。用Hampton试剂盒初筛晶体并用棋盘法进行优化,获得晶形完美、衍射能力很强的hDHODH蛋白复合物单晶。用X射线衍射晶体,用CCP4、Coot软件解析结构,获得hDHODH蛋白复合物晶体结构。从解析的结构中可以看出,抑制剂与蛋白质的吻合度非常高,且抑制剂通过亲水的羧基端与蛋白质356位和147位的酪氨酸形成氢键网络。抑制剂的5元环与蛋白质359位的亮氨酸和360位的苏氨酸相互作用,使抑制剂与蛋白质牢固结合。该复合物晶体结构的顺利解析,将为开发新型特异性抗类风湿性关节炎药物提供重要基础。
Human dihydroorotate dehydrogenase(hDHODH)is a key enzyme that catalyzes the de novo pyrimidine biosynthesis. Recent studies have shown that inhibition of the enzyme can relieve the symptoms of rheumatoid arthritis. Currently there are few inhibitors of the enzyme, so it is of great significance to find an efficient inhibitor of the enzyme. In this study, the hDHODH gene was amplified by PCR. The recombinant plasmid pET-19 b-hDHODH was constructed and expressed in Escherichia coli(E.coli) BL21(DE3) to produce soluble proteins. The protein was purified by Ni~(2+)-NTA affinity chromatography to obtain high purity(90%) hDHODH proteins. The hDHODH protein and inhibitor 3-(5-(ethylthio)-1 H-1,2,4-triazol-3-yl) benzoic acid were mixed and incubated with substrate DHO. The crystal was screened by the Hampton kit and optimized by the chessboard method to obtain a hDHODH protein co-crystal with a perfect crystal shape and strong diffraction ability. The crystal structure was studied by X-ray diffraction, and the structure were analyzed by CCP4 and Coot softwares. Based on the structural analysis, it can be seen that the inhibitor has a very high affinity with protein. The inhibitor formed a hydrogen bond network with the protein at Tyr356 and Tyr147 through the hydrophilic carboxyl terminus and the five-membered ring of the inhibitor interacted with Leu359 and Thr360 of the protein, making the inhibitor firmly bind to the protein. The structural analysis of hDHODH complex crystallization provided an important basis for the development of new specific anti-rheumatoid arthritis drugs.
Human dihydroorotate dehydrogenase(hDHODH)is a key enzyme that catalyzes the de novo pyrimidine biosynthesis. Recent studies have shown that inhibition of the enzyme can relieve the symptoms of rheumatoid arthritis. Currently there are few inhibitors of the enzyme, so it is of great significance to find an efficient inhibitor of the enzyme. In this study, the hDHODH gene was amplified by PCR. The recombinant plasmid pET-19 b-hDHODH was constructed and expressed in Escherichia coli(E.coli) BL21(DE3) to produce soluble proteins. The protein was purified by Ni~(2+)-NTA affinity chromatography to obtain high purity(90%) hDHODH proteins. The hDHODH protein and inhibitor 3-(5-(ethylthio)-1 H-1,2,4-triazol-3-yl) benzoic acid were mixed and incubated with substrate DHO. The crystal was screened by the Hampton kit and optimized by the chessboard method to obtain a hDHODH protein co-crystal with a perfect crystal shape and strong diffraction ability. The crystal structure was studied by X-ray diffraction, and the structure were analyzed by CCP4 and Coot softwares. Based on the structural analysis, it can be seen that the inhibitor has a very high affinity with protein. The inhibitor formed a hydrogen bond network with the protein at Tyr356 and Tyr147 through the hydrophilic carboxyl terminus and the five-membered ring of the inhibitor interacted with Leu359 and Thr360 of the protein, making the inhibitor firmly bind to the protein. The structural analysis of hDHODH complex crystallization provided an important basis for the development of new specific anti-rheumatoid arthritis drugs.
引文
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[14] Powell HR.The rossmann fourier autoindexing algorithm in MOSFLM[J].Acta Crystallogr D Biol Crystallogr,1999,55(Pt 10):1690-1695
[15] Collaborative Computational Project,Number 4..The CCP4 suite:programs for protein crystallography[J].Acta Crystallogr D Biol Crystallogr,1994,50(Pt 5):760-763
[16] Potterton E,Briggs P,Turkenburg M,et al.A graphical user interface to the CCP4 program suite[J].Acta Crystallogr D Biol Crystallogr,2003,59(Pt 7):1131-1137
[17] Murshudov GN,Vagin AA,Dodson EJ.Refinement of macromolecular structures by the maximum- likelihood method[J].Acta Crystallogr D Biol Crystallogr,1997,53(Pt 3):240-255
[18] Bedingfield PT,Cowen D,Acklam P,et al.Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases[J].J Med Chem,2012,55(12):5841-5850
[19] Lewis TA,Sykes DB,Law JM,et al.Development of ML390:a human DHODH inhibitor that induces differentiation in acute myeloid leukemia[J].ACS Med Chem Lett,2016,7(12):1112-1117
[20] Madak JT,Cuthbertson CR,Miyata Y,et al.Design,synthesis,and biological evaluation of 4-Quinoline carboxylic acids as inhibitors of dihydroorotate dehydrogenase[J].J Med Chem,2018,61(12):5162-5186
[21] Fritzson I,Svensson B,Al-karadaghi S,et al.Inhibition of human DHODH by 4-hydroxycoumarins,fenamic acids,and N-(alkylcarbonyl)anthranilic acids identified by structure-guided fragment selection[J].ChemMedChem,2010,5(4):608-617
[22] Ladds MJGW,van Leeuwen IMM,Drummond CJ,et al.A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage[J].Nat Commun,2018,9(1):1107
[23] Hurt DE,Sutton AE,Clardy J.Brequinar derivatives and species-specific drug design for dihydroorotate dehydrogenase[J].Bioorg Med Chem Lett,2006,16(6):1610-1615
[24] Sainas S,Pippione AC,Lupino E,et al.Targeting myeloid differentiation using potent 2-hydroxypyrazolo[1,5-a]pyridine scaffold-based human dihydroorotate dehydrogenase inhibitors[J].J Med Chem,2018,61 (14):6034-6055
[2] Liu S,Neidhardt EA,Grossman TH,et al.Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents[J].Structure,2000,8(1):25-33
[3] Swaminathan P,Saleena L.Evaluation of cardiospermum halicacabum leaf compounds against human DihydroOrotate Dehydrogenase:a target for rheumatoid arthritis using structure based drug designing[J].J Appl Pharm Sci,2017,7(8):48-61
[4] Sitwala ND,Vyas VK,Variya BC,et al.Liquid phase combinatorial synthesis of 1,2,5-trisubstituted benzimidazole derivatives as human DHODH inhibitors[J].Bioorg Chem,2017,75:118-126
[5] Lu K,Cai L,Zhang X,et al.Design,synthesis,and biological evaluation of novel substituted benzamide derivatives bearing a 1,2,3-triazole moiety as potent human dihydroorotate dehydrogenase inhibitors[J].Bioorg Chem,2018,76:528-537
[6] Sainas S,Pippione AC,Giorgis M,et al.Design,synthesis,biological evaluation and X-ray structural studies of potent human dihydroorotate dehydrogenase inhibitors based on hydroxylated azole scaffolds[J].Eur J Med Chem,2017,129:287-302
[7] Vyas VK,Variya B,Ghate MD.Design,synthesis and pharmacological evaluation of novel substituted quinoline-2-carboxamide derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents[J].Eur J Med Chem,2014,82:385-393
[8] Shen W,Ren X,Zhu J,et al.Discovery of a new structural class of competitive hDHODH inhibitors with in vitro and in vivo anti-inflammatory,immunosuppressive effects[J].Eur J Pharmacol,2016,791:205-212
[9] Song W,Li SL,Tong Y,et al.Structure-based design of potent human dihydroorotate dehydrogenase inhibitors as anticancer agents[J].Med Chem Commun,2016,7:1441-1448
[10] Wang JW,Diao YY,Zhu JS,et al.Biological evaluation of quinoline derivatives as inhibitors of human dihydroorotate dehydrogenase[J].Med Chem Commun,2016,7(5):853-858
[11] He D,Xu Z,Dong S,et al.Teriflunomide for multiple sclerosis[J].Cochrane Database Syst Rev,2012,12:CD009882
[12] Gong YH,Liu L,Qi TT,et al.Design,synthesis and structure-activity relationship studies of human dihydroorotate dehydrogenase inhibitors[J].Yao Xue Xue Bao,2017,52(2):264-270
[13] White RM,Cech J,Ratanasirintrawoot S,et al.DHODH modulates transcriptional elongation in the neural crest and melanoma[J].Nature,2011,471(7339):518-522
[14] Powell HR.The rossmann fourier autoindexing algorithm in MOSFLM[J].Acta Crystallogr D Biol Crystallogr,1999,55(Pt 10):1690-1695
[15] Collaborative Computational Project,Number 4..The CCP4 suite:programs for protein crystallography[J].Acta Crystallogr D Biol Crystallogr,1994,50(Pt 5):760-763
[16] Potterton E,Briggs P,Turkenburg M,et al.A graphical user interface to the CCP4 program suite[J].Acta Crystallogr D Biol Crystallogr,2003,59(Pt 7):1131-1137
[17] Murshudov GN,Vagin AA,Dodson EJ.Refinement of macromolecular structures by the maximum- likelihood method[J].Acta Crystallogr D Biol Crystallogr,1997,53(Pt 3):240-255
[18] Bedingfield PT,Cowen D,Acklam P,et al.Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases[J].J Med Chem,2012,55(12):5841-5850
[19] Lewis TA,Sykes DB,Law JM,et al.Development of ML390:a human DHODH inhibitor that induces differentiation in acute myeloid leukemia[J].ACS Med Chem Lett,2016,7(12):1112-1117
[20] Madak JT,Cuthbertson CR,Miyata Y,et al.Design,synthesis,and biological evaluation of 4-Quinoline carboxylic acids as inhibitors of dihydroorotate dehydrogenase[J].J Med Chem,2018,61(12):5162-5186
[21] Fritzson I,Svensson B,Al-karadaghi S,et al.Inhibition of human DHODH by 4-hydroxycoumarins,fenamic acids,and N-(alkylcarbonyl)anthranilic acids identified by structure-guided fragment selection[J].ChemMedChem,2010,5(4):608-617
[22] Ladds MJGW,van Leeuwen IMM,Drummond CJ,et al.A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage[J].Nat Commun,2018,9(1):1107
[23] Hurt DE,Sutton AE,Clardy J.Brequinar derivatives and species-specific drug design for dihydroorotate dehydrogenase[J].Bioorg Med Chem Lett,2006,16(6):1610-1615
[24] Sainas S,Pippione AC,Lupino E,et al.Targeting myeloid differentiation using potent 2-hydroxypyrazolo[1,5-a]pyridine scaffold-based human dihydroorotate dehydrogenase inhibitors[J].J Med Chem,2018,61 (14):6034-6055