Virus nucleoprotein (NP) is an emerging target for drug development for Influenza. We designed benzamide derivatives as new inhibitors of NP that demonstrate good potency in blocking influenza A. Screening revealed th...Virus nucleoprotein (NP) is an emerging target for drug development for Influenza. We designed benzamide derivatives as new inhibitors of NP that demonstrate good potency in blocking influenza A. Screening revealed that compound 39 was the most potent molecule in the series, exhibiting IC<sub>50</sub> values of 0.46 and 0.27 μM in blocking the replication of H3N2 (A/HK/8/68) and (A/WSN/33) influenza A viral strains. The observed inhibition of viral replication correlated well with cytopathic protection. Furthermore, based on computational analysis and fluorescence microscopy, it was determined that compound 39 inhibited nuclear accumulation by targeting influenza A viral nucleoproteins. Finally, the rodent pharmacokinetic profile of compound 32 displayed half-life of greater than 4 hours and bioavailability greater than 20%, suggesting this class of molecules had drug-like properties.展开更多
甲型流感病毒(influenza A virus,IAV)严重威胁人类健康。病毒核心是核糖核蛋白复合物(viral ribonucleoprotein complexes,vRNPs),是病毒基因组转录和复制的最小功能单位。核蛋白(nucleoprotein,NP)是vRNP的重要组成部分之一,在流感病...甲型流感病毒(influenza A virus,IAV)严重威胁人类健康。病毒核心是核糖核蛋白复合物(viral ribonucleoprotein complexes,vRNPs),是病毒基因组转录和复制的最小功能单位。核蛋白(nucleoprotein,NP)是vRNP的重要组成部分之一,在流感病毒整个生命周期起着关键作用。机体存在多种宿主因子通过NP影响病毒增殖。本文就通过NP影响vRNP核输入、病毒基因组转录和复制、vRNP核输出以及vRNP组装这几个方面的宿主因子进行综述,以期为治疗IAV新靶点的发现及新药物的研发提供参考。展开更多
目的构建以乙型肝炎病毒核心蛋白(Hepatitis B virus coreprotein,HBc)颗粒为载体的含流感病毒M2基质蛋白胞外功能区(M2 ectodomain,M2e)和核蛋白(Nucleoprotein,NP)保守表位的流感通用疫苗,评价其抗不同亚型流感病毒感染的保护作用。...目的构建以乙型肝炎病毒核心蛋白(Hepatitis B virus coreprotein,HBc)颗粒为载体的含流感病毒M2基质蛋白胞外功能区(M2 ectodomain,M2e)和核蛋白(Nucleoprotein,NP)保守表位的流感通用疫苗,评价其抗不同亚型流感病毒感染的保护作用。方法采用基因工程方法将流感病毒M2e的3拷贝重复片段与NP的CTL表位串联,插入HBc刺突顶端的免疫优势决定区,构建重组表达质粒pET-21a-HBc-3M2e-NP,转化大肠杆菌BL21(DE3),IPTG16℃低温诱导表达。表达的融合蛋白HBc-3M2e-NP经纯化后,电镜观察病毒样颗粒形成情况。纯化的融合蛋白分别经鼻腔和腹腔免疫小鼠,对照组注射等体积的PBS,间接ELISA法检测小鼠血清IgG抗体水平;流式细胞术检测小鼠脾组织中CD4+、CD8+T淋巴细胞水平;以流感病毒攻毒,检测融合蛋白的保护效果。结果重组表达质粒pET-21a-HBc-3M2e-NP经PCR及双酶切鉴定证明构建正确。表达的融合蛋白相对分子质量约为29000,以包涵体和可溶性两种形式表达,且可与鼠抗M2e单抗发生特异性反应。纯化的融合蛋白纯度大于90%,且能够自动装配成病毒样颗粒。用该病毒样颗粒免疫小鼠,可诱导小鼠产生针对不同流感病毒毒株的特异性抗体;2种途径免疫的小鼠脾组织中CD4+T淋巴细胞含量和CD4+/CD8+T淋巴细胞比例均较对照组明显升高;攻毒试验结果显示,具有交叉保护作用。结论构建的流感通用疫苗能够诱导机体产生高水平的体液免疫和细胞免疫应答及有效的交叉保护作用,为流感通用疫苗的深入研究奠定了基础。展开更多
文摘Virus nucleoprotein (NP) is an emerging target for drug development for Influenza. We designed benzamide derivatives as new inhibitors of NP that demonstrate good potency in blocking influenza A. Screening revealed that compound 39 was the most potent molecule in the series, exhibiting IC<sub>50</sub> values of 0.46 and 0.27 μM in blocking the replication of H3N2 (A/HK/8/68) and (A/WSN/33) influenza A viral strains. The observed inhibition of viral replication correlated well with cytopathic protection. Furthermore, based on computational analysis and fluorescence microscopy, it was determined that compound 39 inhibited nuclear accumulation by targeting influenza A viral nucleoproteins. Finally, the rodent pharmacokinetic profile of compound 32 displayed half-life of greater than 4 hours and bioavailability greater than 20%, suggesting this class of molecules had drug-like properties.
文摘甲型流感病毒(influenza A virus,IAV)严重威胁人类健康。病毒核心是核糖核蛋白复合物(viral ribonucleoprotein complexes,vRNPs),是病毒基因组转录和复制的最小功能单位。核蛋白(nucleoprotein,NP)是vRNP的重要组成部分之一,在流感病毒整个生命周期起着关键作用。机体存在多种宿主因子通过NP影响病毒增殖。本文就通过NP影响vRNP核输入、病毒基因组转录和复制、vRNP核输出以及vRNP组装这几个方面的宿主因子进行综述,以期为治疗IAV新靶点的发现及新药物的研发提供参考。
文摘目的构建以乙型肝炎病毒核心蛋白(Hepatitis B virus coreprotein,HBc)颗粒为载体的含流感病毒M2基质蛋白胞外功能区(M2 ectodomain,M2e)和核蛋白(Nucleoprotein,NP)保守表位的流感通用疫苗,评价其抗不同亚型流感病毒感染的保护作用。方法采用基因工程方法将流感病毒M2e的3拷贝重复片段与NP的CTL表位串联,插入HBc刺突顶端的免疫优势决定区,构建重组表达质粒pET-21a-HBc-3M2e-NP,转化大肠杆菌BL21(DE3),IPTG16℃低温诱导表达。表达的融合蛋白HBc-3M2e-NP经纯化后,电镜观察病毒样颗粒形成情况。纯化的融合蛋白分别经鼻腔和腹腔免疫小鼠,对照组注射等体积的PBS,间接ELISA法检测小鼠血清IgG抗体水平;流式细胞术检测小鼠脾组织中CD4+、CD8+T淋巴细胞水平;以流感病毒攻毒,检测融合蛋白的保护效果。结果重组表达质粒pET-21a-HBc-3M2e-NP经PCR及双酶切鉴定证明构建正确。表达的融合蛋白相对分子质量约为29000,以包涵体和可溶性两种形式表达,且可与鼠抗M2e单抗发生特异性反应。纯化的融合蛋白纯度大于90%,且能够自动装配成病毒样颗粒。用该病毒样颗粒免疫小鼠,可诱导小鼠产生针对不同流感病毒毒株的特异性抗体;2种途径免疫的小鼠脾组织中CD4+T淋巴细胞含量和CD4+/CD8+T淋巴细胞比例均较对照组明显升高;攻毒试验结果显示,具有交叉保护作用。结论构建的流感通用疫苗能够诱导机体产生高水平的体液免疫和细胞免疫应答及有效的交叉保护作用,为流感通用疫苗的深入研究奠定了基础。
