Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in D...Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in DENV replication because of its function in cleavage of the viral polyprotein;thus,it is considered a promising target for antiviral discovery.In this study,we developed a high-throughput screening system based on the NS2B-NS3 protease to identify candidates from an FDA-approved drug library.Eltrombopag was screened out of 3273 drugs,and demonstrated inhibition on DENV 2 at the micromolar level in vitro,significantly reducing viral loads in the targeted organs of challenged mice following intraperitoneal injection.Further mechanistic analysis showed that eltrombopag allosterically binds to the DENV 2 NS2B-NS3 protease in a reversible,noncompetitive manner,therefore inhibiting DENV 2 at the post-infection stage.In addition,eltrombopag inhibited the NS2B-NS3 proteases of DENV 4 and Zika virus,suggesting its potential as a broadspectrum antiviral agent.This study repurposed eltrombopag as a promising antiviral agent against DENV,providing an alternative for antiviral development against flaviviruses.展开更多
应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p Q...应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p QENS2转化 JM10 9菌株 ,诱导表达出 N端含有 6个 His的全长 NS2蛋白 ,用 Ni-NTA- agarose柱层析纯化 ,获得提纯的全长 NS2蛋白 .展开更多
基金supported by the National Natural Science Foundation of China(Grant No.82130101)the Youth Innovation Promotion Association of CAS(Grant No.2021333).
文摘Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in DENV replication because of its function in cleavage of the viral polyprotein;thus,it is considered a promising target for antiviral discovery.In this study,we developed a high-throughput screening system based on the NS2B-NS3 protease to identify candidates from an FDA-approved drug library.Eltrombopag was screened out of 3273 drugs,and demonstrated inhibition on DENV 2 at the micromolar level in vitro,significantly reducing viral loads in the targeted organs of challenged mice following intraperitoneal injection.Further mechanistic analysis showed that eltrombopag allosterically binds to the DENV 2 NS2B-NS3 protease in a reversible,noncompetitive manner,therefore inhibiting DENV 2 at the post-infection stage.In addition,eltrombopag inhibited the NS2B-NS3 proteases of DENV 4 and Zika virus,suggesting its potential as a broadspectrum antiviral agent.This study repurposed eltrombopag as a promising antiviral agent against DENV,providing an alternative for antiviral development against flaviviruses.
