| 129 | 0 | 163 |
| 下载次数 | 被引频次 | 阅读次数 |
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。
Abstract：Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus found primarily in Siberian(Russia). It infects humans through tick bites or contact with infectious materials. Symptoms of infection include headache, cough, fever, and bleeding. Specific drugs targeting this virus are not available. The translated genome of OHFV forms polyproteins, among which the NS3 protease cleaves polyproteins to fulfill their respective roles in the viral replication cycle. The hydrophilic region of NS2B acts as a cofactor to maintain the hydrolytic activity of NS3 protease, having an indispensable role in the viral lifecycle. We aimed to develop small-molecule inhibitors of Omsk hemorrhagic fever by targeting the NS2B-NS3 protease. The NS2B-NS3 of OHFV protease was expressed using the Escherichia coli expression system to obtain highly pure and homogeneous protein. Simultaneously, a drug screening system was established to conduct inhibitor screening.The final drug-screening system consisted of Tris(20 mmol/L) and 20% glycerol at pH 8.5, with a final protein concentration of 2 μmol/L and a substrate concentration of 100 μmol/L. Utilizing this system for inhibitor screening, the compound zinc pyrithione, was identified with percent inhibition up to 99% against NS2B-NS3 protease of OHFV. The half-maximal inhibitory concentration of this inhibitor was determined, and its inhibitory type was identified as reversible. Zinc pyrithione shows promise to be a lead compound against Omsk hemorrhagic fever virus, providing a research basis for drug development targeting this virus.
[1] Shah SZ, Jabbar B, Ahmed N, et al. Epidemiology,pathogenesis, and control of a tick-borne diseasekyasanur forest disease:current status and future directions[J]. Front Cell Infect Microbiol, 2018, 8:149. DOI:10. 3389/fcimb. 2018. 00149.
[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~~