2024 05 v.40 986-995
鄂木斯克出血热病毒NS2B-NS3蛋白酶药物筛选体系的建立及抑制剂筛选
基金项目(Foundation):
国家自然科学基金面上项目（项目号：8217033959）,题目：功能未知小囊泡在膀胱血-尿屏障形成中的作用和机制研究;国家自然科学基金青年项目（项目号：8220082920）,题目：Wolbachia诱导胞质不相容性表型多样性分子机制研究~~
邮箱(Email):
zefang-wang@163.com;yun-jiexiao2016@tju.edu.cn;
DOI:
10.13242/j.cnki.bingduxuebao.004559
中文作者单位:
天津大学生命科学学院;上海科技大学生命科学与技术学院;
摘要(Abstract):
鄂木斯克出血热病毒（Omsk hemorrhagic fever virus,OHFV）是一种蜱传黄病毒，主要分布在俄罗斯西伯利亚地区，通过蜱虫叮咬、接触感染物等途径感染人类。感染后症状包括头痛、咳嗽、发烧并伴有出血。目前尚无针对该病毒的特效药物。鄂木斯克出血热病毒基因组经翻译后形成多聚蛋白，其中NS3蛋白酶可将多聚蛋白切割从而发挥其各自在病毒复制周期中的作用。NS2B的亲水区可以作为NS3蛋白酶的辅因子维持其水解活性，在该病毒的生命周期中发挥着不可或缺的作用。该文旨在以NS2B-NS3蛋白酶为靶点开发有效抑制鄂木斯克出血热的小分子抑制剂。选用大肠杆菌表达系统表达鄂木斯克出血热病毒NS2B-NS3蛋白酶，以获得纯度高且性质均一的蛋白。同时建立药物筛选体系，开展抑制剂筛选。最终建立的药物筛选体系为20 mmol/L Tris,20%甘油，pH 8.5，蛋白终浓度为2μmol/L，底物终浓度为100μmol/L。利用该体系进行抑制剂筛选，筛选出对OHFV NS2B-NS3蛋白酶抑制率高达99%的化合物吡啶硫酮锌，并对该抑制剂进行了IC50的测定和抑制剂类型的判定，其为可逆抑制剂。吡啶硫酮锌有望成为抗鄂木斯克出血热病毒的先导化合物，为针对该病毒今后的药物开发提供研究基础。
关键词(KeyWords):
鄂木斯克出血热;;黄病毒;;NS2B-NS3蛋白酶;;蛋白酶抑制剂
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参考文献
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[15]Murray CL, Jones CT, Rice CM. Architects of assembly:roles of Flaviviridae non-structural proteins in virion morphogenesis[J]. Nat Rev Microbiol, 2008, 6:699-708. DOI:10. 1038/nrmicro1928.
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[19]Noble CG, Shi PY. Structural biology of dengue virus enzymes:Towards rational design of therapeutics[J].Antivir Res, 2012, 96(2):115-126. DOI:10. 1016/j.antiviral. 2012. 09. 007.
[20]Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery[J]. Antivir Res, 2009, 81(1):6-15. DOI:10. 1016/j. antiviral. 2008. 08. 004.
[21]Lescar J, Luo D, Xu T, et al. Towards the design of antiviral inhibitors against flaviviruses:The case for the multifunctional NS3 protein from Dengue virus as a target[J]. Antivir Res, 2008, 80(2):94-101. DOI:10. 1016/j. antiviral. 2008. 07. 001.
[22]Davidson AD. Chapter 2. New insights into flavivirus nonstructural protein 5[J]. Adv Virus Res, 2009, 74:41-101. DOI:10. 1016/s0065-3527(09)74002-3.
[23]Chambers TJ, McCourt DW, Rice CM. Production of yellow fever virus proteins in infected cells:Identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera[J]. Virology, 1990, 177(1):159-174. DOI:10. 1016/0042-6822(90)90470-C.
[24]Li J, Lim SP, Beer D, et al. Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries[J]. J Biol Chem, 2005, 280(31):28766-28774. DOI:10. 1074/jbc. M500588200.
[25]Yusof R, Clum S, Wetzel M, et al. Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro[J]. J Biol Chem,2000, 275(14):9963-9969. DOI:10. 1074/jbc. 275. 14. 9963.
[26]Zhang L, Mohan PM, Padmanabhan R. Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5[J]. J Virol, 1992, 66(12):7549-7554. DOI:10. 1128/jvi. 66. 12. 7549-7554. 1992.
[27]Clum S, Ebner KE, Padmanabhan R. Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(pro)of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B[J]. J Biol Chem, 1997,272(49):30715-30723. DOI:10. 1074/jbc. 272. 49. 30715.
[28]Wu CF, Wang SH, Sun CM, et al. Activation of dengue protease autocleavage at the NS2B–NS3junction by recombinant NS3 and GST–NS2B fusion proteins[J]. J Virol Meth, 2003, 114(1):45-54.DOI:10. 1016/j. jviromet. 2003. 09. 001.
[29]Phong WY, Moreland NJ, Lim SP, et al. Dengue protease activity:the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target[J]. Biosci Rep, 2011, 31(5):399-409. DOI:10. 1042/bsr20100142.
[30]刘晓丽，闫干干，闫浩浩，等.登革病毒NS2B-NS3蛋白酶在大肠埃希菌中表达条件的优化及酶活性测定[J].中国生物制品学杂志，2023, 36(11):1306-1312.DOI:10. 13200/j. cnki. cjb. 003855.
[31]Akaberi D, B?hlstr?m A, Chinthakindi PK, et al.Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors[J]. Antivir Res, 2021, 190:105074. DOI:10. 1016/j. antiviral. 2021. 105074.
[32]Gawish R, Starkl P, Pimenov L, et al. ACE2 is the critical in vivo receptor for SARS-CoV-2 in a novel COVID-19 mouse model with TNF-and IFNγ-driven immunopathology[J]. Elife, 2022, 11:e74623. DOI:10. 7554/elife. 74623.
[33]Mayor-Ibarguren A, Busca-Arenzana C, RoblesMarhuendaá. A hypothesis for the possible role of zinc in the immunological pathways related to COVID-19infection[J]. Front Immunol, 2020, 11:1736. DOI:10. 3389/fimmu. 2020. 01736.
[34]Pormohammad A, Monych NK, Turner RJ. Zinc and SARS-CoV-2:a molecular modeling study of Zn interactions with RNA-dependent RNA-polymerase and3C-like proteinase enzymes[J]. Int J Mol Med, 2021,47(1):326-334. DOI:10. 3892/ijmm. 2020. 4790.
[35]Grifagni D, Calderone V, Giuntini S, et al. SARS-CoV-2 Mpro inhibition by a zinc ion:structural features and hints for drug design[J]. Chem Commun, 2021, 57(64):7910-7913. DOI:10. 1039/D1CC02956H.
[36]Kladnik J, Dolinar A, Kljun J, et al. Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication[J]. J Enzyme Inhib Med Chem, 2022, 37(1):2158-2168. DOI:10. 1080/14756366. 2022. 2108417.
[37]de Arruda EGR, Rocha BA, Barrionuevo MVF, et al.The influence of ZnII coordination sphere and chemical structure over the reactivity of metallo-β-lactamase model compounds[J]. Dalton Trans, 2019, 48(9):2900-2916. DOI:10. 1039/C8DT03905D.
[2] Im JH, Baek JH, Durey A, et al. Geographic distribution of Tick-borne encephalitis virus complex[J]. J Vector Borne Dis, 2020, 57(1):14-22. DOI:10. 4103/0972-9062. 308794.
[3] Brecher M, Li Z, Liu B, et al. A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease[J/OL]. PLoS Pathog, 2017, 13(5):e1006411. DOI:10. 1371/journal. ppat. 1006411.
[4] Kovalev SY, Mazurina EA. Omsk hemorrhagic fever virus is a tick-borne encephalitis virus adapted to muskrat through host-jumping[J]. J Med Virol, 2022, 94(6):2510-2518. DOI:10. 1002/jmv. 27581.
[5] R??ek D, Yakimenko VV, Karan LS, et al. Omsk haemorrhagic fever[J]. Lancet, 2010, 376(9758):2104-2113. DOI:10. 1016/s0140-6736(10)61120-8.
[6] Kovalev SY, Mazurina EA, Yakimenko VV.Molecular variability and genetic structure of Omsk hemorrhagic fever virus, based on analysis of the complete genome sequences[J]. Ticks Tick Borne Dis,2021, 12(2):101627. DOI:10. 1016/j.ttbdis. 2020. 101627.
[7] Lin D, Li L, Dick D, et al. Analysis of the complete genome of the tick-borne flavivirus Omsk hemorrhagic fever virus[J]. Virology, 2003, 313(1):81-90. DOI:10. 1016/S0042-6822(03)00246-0.
[8] Alvarez DE, Lodeiro MF, Filomatori CV, et al.Structural and functional analysis of dengue virus RNA[J]. Novartis Found Symp, 2006, 277:120-32;discussion132-5, 251-3.
[9] Filomatori CV, Lodeiro MF, Alvarez DE, et al. A 5'RNA element promotes dengue virus RNA synthesis on a circular genome[J]. Genes Dev, 2006, 20(16):2238-2249. DOI:10. 1101/gad. 1444206.
[10]Khromykh AA, Kondratieva N, Sgro JY, et al.Significance in replication of the terminal nucleotides of the flavivirus genome[J]. J Virol, 2003, 77(19):10623-10629. DOI:10. 1128/jvi. 77. 19. 10623-10629. 2003.
[11]Markoff L. 5'-and 3'-noncoding regions in flavivirus RNA[J]. Adv Virus Res, 2003, 59:177-228. DOI:10. 1016/s0065-3527(03)59006-6.
[12]Xu T, Sampath A, Chao A, et al. Structure of the Dengue virus helicase/nucleoside triphosphatase catalytic domain at a resolution of 2. 4 A[J]. J Virol,2005, 79(16):10278-10288. DOI:10. 1128/jvi. 79. 16. 10278-10288. 2005.
[13]Yap TL, Xu T, Chen YL, et al. Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1. 85-angstrom resolution[J]. J Virol, 2007, 81(9):4753-4765. DOI:10. 1128/jvi. 02283-06.
[14]Malet H, MasséN, Selisko B, et al. The flavivirus polymerase as a target for drug discovery[J]. Antivir Res, 2008, 80(1):23-35. DOI:10. 1016/j.antiviral. 2008. 06. 007.
[15]Murray CL, Jones CT, Rice CM. Architects of assembly:roles of Flaviviridae non-structural proteins in virion morphogenesis[J]. Nat Rev Microbiol, 2008, 6:699-708. DOI:10. 1038/nrmicro1928.
[16]Paul D, Bartenschlager R. Architecture and biogenesis of plus-strand RNA virus replication factories[J]. World J Virol, 2013, 2(2):32-48. DOI:10. 5501/wjv. v2.i2. 32.
[17]Bollati M, Alvarez K, Assenberg R, et al. Structure and functionality in flavivirus NS-proteins:Perspectives for drug design[J]. Antivir Res, 2010, 87(2):125-148. DOI:10. 1016/j. antiviral. 2009. 11. 009.
[18]Lim SP, Wang QY, Noble CG, et al. Ten years of dengue drug discovery:Progress and prospects[J].Antivir Res, 2013, 100(2):500-519. DOI:10. 1016/j.antiviral. 2013. 09. 013.
[19]Noble CG, Shi PY. Structural biology of dengue virus enzymes:Towards rational design of therapeutics[J].Antivir Res, 2012, 96(2):115-126. DOI:10. 1016/j.antiviral. 2012. 09. 007.
[20]Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery[J]. Antivir Res, 2009, 81(1):6-15. DOI:10. 1016/j. antiviral. 2008. 08. 004.
[21]Lescar J, Luo D, Xu T, et al. Towards the design of antiviral inhibitors against flaviviruses:The case for the multifunctional NS3 protein from Dengue virus as a target[J]. Antivir Res, 2008, 80(2):94-101. DOI:10. 1016/j. antiviral. 2008. 07. 001.
[22]Davidson AD. Chapter 2. New insights into flavivirus nonstructural protein 5[J]. Adv Virus Res, 2009, 74:41-101. DOI:10. 1016/s0065-3527(09)74002-3.
[23]Chambers TJ, McCourt DW, Rice CM. Production of yellow fever virus proteins in infected cells:Identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera[J]. Virology, 1990, 177(1):159-174. DOI:10. 1016/0042-6822(90)90470-C.
[24]Li J, Lim SP, Beer D, et al. Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries[J]. J Biol Chem, 2005, 280(31):28766-28774. DOI:10. 1074/jbc. M500588200.
[25]Yusof R, Clum S, Wetzel M, et al. Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro[J]. J Biol Chem,2000, 275(14):9963-9969. DOI:10. 1074/jbc. 275. 14. 9963.
[26]Zhang L, Mohan PM, Padmanabhan R. Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5[J]. J Virol, 1992, 66(12):7549-7554. DOI:10. 1128/jvi. 66. 12. 7549-7554. 1992.
[27]Clum S, Ebner KE, Padmanabhan R. Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(pro)of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B[J]. J Biol Chem, 1997,272(49):30715-30723. DOI:10. 1074/jbc. 272. 49. 30715.
[28]Wu CF, Wang SH, Sun CM, et al. Activation of dengue protease autocleavage at the NS2B–NS3junction by recombinant NS3 and GST–NS2B fusion proteins[J]. J Virol Meth, 2003, 114(1):45-54.DOI:10. 1016/j. jviromet. 2003. 09. 001.
[29]Phong WY, Moreland NJ, Lim SP, et al. Dengue protease activity:the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target[J]. Biosci Rep, 2011, 31(5):399-409. DOI:10. 1042/bsr20100142.
[30]刘晓丽，闫干干，闫浩浩，等.登革病毒NS2B-NS3蛋白酶在大肠埃希菌中表达条件的优化及酶活性测定[J].中国生物制品学杂志，2023, 36(11):1306-1312.DOI:10. 13200/j. cnki. cjb. 003855.
[31]Akaberi D, B?hlstr?m A, Chinthakindi PK, et al.Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors[J]. Antivir Res, 2021, 190:105074. DOI:10. 1016/j. antiviral. 2021. 105074.
[32]Gawish R, Starkl P, Pimenov L, et al. ACE2 is the critical in vivo receptor for SARS-CoV-2 in a novel COVID-19 mouse model with TNF-and IFNγ-driven immunopathology[J]. Elife, 2022, 11:e74623. DOI:10. 7554/elife. 74623.
[33]Mayor-Ibarguren A, Busca-Arenzana C, RoblesMarhuendaá. A hypothesis for the possible role of zinc in the immunological pathways related to COVID-19infection[J]. Front Immunol, 2020, 11:1736. DOI:10. 3389/fimmu. 2020. 01736.
[34]Pormohammad A, Monych NK, Turner RJ. Zinc and SARS-CoV-2:a molecular modeling study of Zn interactions with RNA-dependent RNA-polymerase and3C-like proteinase enzymes[J]. Int J Mol Med, 2021,47(1):326-334. DOI:10. 3892/ijmm. 2020. 4790.
[35]Grifagni D, Calderone V, Giuntini S, et al. SARS-CoV-2 Mpro inhibition by a zinc ion:structural features and hints for drug design[J]. Chem Commun, 2021, 57(64):7910-7913. DOI:10. 1039/D1CC02956H.
[36]Kladnik J, Dolinar A, Kljun J, et al. Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication[J]. J Enzyme Inhib Med Chem, 2022, 37(1):2158-2168. DOI:10. 1080/14756366. 2022. 2108417.
[37]de Arruda EGR, Rocha BA, Barrionuevo MVF, et al.The influence of ZnII coordination sphere and chemical structure over the reactivity of metallo-β-lactamase model compounds[J]. Dalton Trans, 2019, 48(9):2900-2916. DOI:10. 1039/C8DT03905D.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004559
中图分类号:R914
引用信息:
[1]陈雅丽,封守琴,杨海涛等.鄂木斯克出血热病毒NS2B-NS3蛋白酶药物筛选体系的建立及抑制剂筛选[J].病毒学报,2024,40(05):986-995.DOI:10.13242/j.cnki.bingduxuebao.004559.
基金信息:
国家自然科学基金面上项目（项目号：8217033959）,题目：功能未知小囊泡在膀胱血-尿屏障形成中的作用和机制研究;国家自然科学基金青年项目（项目号：8220082920）,题目：Wolbachia诱导胞质不相容性表型多样性分子机制研究~~
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