CN116421606B - 小分子药物amg7703在抗流感病毒感染中的应用 - Google Patents
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Abstract
本发明公开了小分子药物AMG7703在抗流感病毒感染中的应用,实验结果表明,小分子药物AMG7703不仅具有抗低致病性流感病毒复制的作用,还能显著地抑制高致病性流感病毒复制,且靶向流感病毒复制周期的晚期阶段;小分子药物AMG7703是宿主因子FFAR2选择性变构激动剂,进一步夯实了宿主因子FFAR2在调节流感病毒复制过程中发挥重要的调节功能,能够成为理想的抗流感病毒复制的药物靶标。
Description
技术领域
本发明涉及靶向宿主因子FFAR2的小分子药物AMG7703在抗流感病毒感染中的应用,属于生物医药领域。
背景技术
流感病毒是有囊膜的单股负链RNA病毒,其中A型流感病毒的基因组分为8个节段,可编码至少15种蛋白。在流感病毒紧急防控中常用药物有金刚烷胺/利巴韦林(靶向流感病毒M2离子通道),奥赛米韦和帕拉米韦(靶向神经氨酸酶)。然而,药物长时间应用使得耐药性毒株在世界范围内不断出现,例如:2005年从感染H5N1亚型流感病毒患者体内分离得到对奥赛米韦的耐药毒株;2008-2009年美国病毒监测结果表明,具有耐药性突变的H1N1季节性流感病毒比例高达99.4%。除此之外,2018年2月日本批准针对PA内切核酸酶活性的抑制剂Xofluza,但随后的流行病学监测结果显示已经出现了抗该抑制剂的耐药毒株,因而寻找新的药物靶点迫在眉睫。而解决这一毒株耐药性问题的切入点为挖掘靶向具有调节流感病毒复制的宿主因子的小分子药物。
GPCR蛋白家族作为细胞膜表面最大的超家族受体,能够将细胞外信号转化为关键的生理效应,同时与一些危害人类健康的重大疾病(癌症、糖尿病等)密切相关。其众多的下游信号通路使得该家族蛋白在药物研发领域具有巨大价值。类似地,GPCR家族蛋白在调节病毒复制过程中也发挥着重要作用,已有研究报道表明GPCR蛋白参与启动流感病毒、狂犬病毒、埃博拉病毒、马尔堡病毒、艾滋病病毒的内吞过程,这些宿主因子主要涉及游离脂肪酸受体2(FFAR2)、代谢型谷氨酸受体II型(mGLuR2)、组胺受体、5-羟色胺受体(5-HT)、毒蕈碱型乙酰胆碱受体(mAChR)、肾上腺素能受体(AR)和趋化因子受体4(CXCR4)等。其中FFAR2属于视紫红质样受体家族,能够介导许多信号通路:其激活能够结合百日咳毒素敏感的Gi/o蛋白和百日咳毒素不敏感的Gq蛋白,导致肌醇1,4,5-三磷酸形成、细胞内Ca2+释放、ERK1/2激活、cAMP积累抑制以及p38、JunN端蛋白激酶(JNK)和Akt信号通路的调节耦合。小分子药物AMG7703(结构如图1所示)作为FFAR2选择性变构激动剂,其是否可以抑制流感病毒复制尚不明确。
发明内容
本发明所要解决的技术问题为:提供一种小分子化合物AMG7703在抗流感病毒感染中的新用途。
本发明的技术方案为:AMG7703或其药学上可接受的盐或酯在制备抗流感病毒感染的药物中的用途,所述AMG7703的结构如下所示:
进一步地,所述流感病毒为H1N1、H5N1或H9N2亚型流感病毒。
与现有技术相比,本发明具有以下有益效果:
本发明研究证明了小分子药物AMG7703不仅具有抗低致病性流感病毒复制的作用,还能显著地抑制高致病性流感病毒复制,且靶向流感病毒复制周期的晚期阶段,进一步夯实了宿主因子FFAR2在调节流感病毒复制过程中发挥重要的调节功能,能够成为理想的抗流感病毒复制的药物靶标。
附图说明
图1为靶向FFAR2的小分子化合物AMG7703的化学结构式;
图2为利用CellTiter Glo试剂盒测定不同浓度的AMG7703对A549细胞的毒性;
图3为不同浓度的AMG7703抗H1N1亚型流感病毒复制的作用;
图4为AMG7703抑制低剂量H1N1流感病毒多轮复制时各病毒蛋白表达;
图5为AMG7703对vRNP入核无影响;
图6为AMG7703抑制高剂量H1N1流感病毒单轮感染时晚期病毒蛋白的表达;
图7为利用CellTiter Glo试剂盒测定不同浓度的AMG7703对HEK293T细胞的毒性;
图8为不同浓度的AMG7703对三种聚合酶蛋白和NP蛋白的表达无影响;
图9为不同浓度的AMG7703抗甲流H1N1亚型流感病毒复制的作用;
图10为不同浓度的AMG7703抗H5N1亚型流感病毒复制的作用;
图11为不同浓度的AMG7703抗禽源H9N2亚型流感病毒复制的作用。
具体实施方式
实施材料:本实验研究中所使用的材料和耗材,如无特殊指明,均可从商业途径购买得到。A549细胞和HEK293T均购自于ATCC,质粒载体来自于Addgene或本实验室独立构建,小分子化合物AMG7703购自MCE(目录号HY-114011)。H1N1(WSN)、甲流H1N1(FZ09)、H9N2(SC197)和H5N1(AH05)均由本实验室分离并保存。本实验研究中所使用的H5N1病毒在中国农业科学院哈尔滨兽医研究所生物安全3级实验室进行,生物安全3级实验室由农业部和中国合格评定国家认可委员会(CNAS)批准使用。下述实验方法为标准的分子生物学、细胞生物学或病毒学等操作程序,领域内的研究人员可以方便地理解和操作。
一、利用CellTiter Glo试剂盒测定不同浓度的AMG7703对A549细胞的毒性
图1所示的小分子化合物AMG7703溶解于DMSO中,配制成100mM贮存液,分装并冻存于-80℃。
将A549细胞悬液平铺于96孔板中,每孔中加入100μL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成50、40、30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入150μL上述不同浓度的AMG7703工作液(各三孔)于96孔板中,于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在孵育24h后,每孔中加入100μL CellTiter-Glo reagent(购自Promega,目录号G7572)于37℃培养箱裂解10min,荧光数值用GloMax 96Microplate Luminometer(Promega)进行检测。每个处理均设置3个重复孔,每个实验进行至少2次重复。实验结果利用Graphpad Prism 5数据处理软件进行作图,并进行统计分析。
结果如图2所示,与阴性对照组相比,50μM AMG7703能够对A549细胞活力产生接近15%的抑制作用,40μM AMG7703能够对A549细胞活力产生接近8%的抑制作用;30、20μMAMG7703对A549细胞活力无影响。**代表p<0.01,*代表p<0.05。
二、不同浓度的AMG7703抗H1N1亚型流感病毒复制的作用
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成40、30、20μM工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述不同浓度的AMG7703工作液(各三孔)于12孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将H1N1(WSN)病毒以MOI=0.01的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述相应浓度的AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的12和24h时采集细胞上清液,进行蚀斑滴定。
结果如图3所示,40μM AMG7703在12和24h分别能够抑制H1N1(WSN)病毒复制达6.9倍和4.2倍;30μM AMG7703在12和24h分别能够抑制H1N1(WSN)病毒复制达3.4倍和3.7倍;20μM AMG7703在12和24h分别能够抑制H1N1(WSN)病毒复制达2.7倍和2.6倍。
上述实验结果显示,AMG7703在激活FFAR2后能够抑制H1N1(WSN)病毒的复制。
三、AMG7703抑制低剂量H1N1流感病毒多轮复制时病毒蛋白表达
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述30、20μM AMG7703工作液于12孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将H1N1(WSN)病毒以MOI=0.01的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述30、20μM AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的24h时弃掉细胞上清液,用1×loading裂解细胞,通过免疫印迹法进行检测在流感病毒多轮复制过程中AMG7703对病毒蛋白的表达影响,进而明确AMG7703对病毒的抑制作用。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1和NP抗体(本实验室制备单克隆抗体)、M1抗体(GeneTex,目录号GTX125928)、NS1抗体(GeneTex,目录号GTX125990)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight 800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图4所示,30、20μM AMG7703在24h均能够呈剂量依赖性抑制低剂量H1N1(WSN)亚型流感病毒感染时病毒蛋白的表达,尤其对晚期蛋白M1表达抑制效果最明显,进一步夯实了AMG7703对流感病毒复制的抑制作用。
四、AMG7703对vRNP入核无影响
将A549细胞悬液平铺于激光共聚焦小皿中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述30、20μM AMG7703工作液于激光共共聚焦小皿中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将H1N1(WSN)病毒以MOI=5的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述30、20μM AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,分别在病毒感染后的2、3、4h时弃掉细胞上清液,用4%多聚甲醛(购自Solarbio,目录号P1110-500)进行室温固定30min。用PBS洗涤三次,每次室温摇晃10min,用0.5%Triton-X100室温通透15min,用PBS洗涤三次后用5% BSA(PBS溶解)室温封闭1h,而后用针对流感病毒NP蛋白的鼠源单克隆抗体(1:200)室温孵育2h,用PBS洗涤三次后加入Alexa Fluor 633donkey anti-mouse IgG二抗,室温遮光孵育1h,用PBS洗涤3次后加入DAPI细胞核染料(1:500),室温孵育15min,用PBS洗涤三次后用LSM800激光共聚焦显微镜(ZEISS)随机拍摄多个视野,并统计分析至少250个细胞中NP蛋白的入核情况。
激光共聚焦实验中使用NP抗体(本实验室制备鼠单克隆抗体)和Alexa Fluor633donkey anti-mouse IgG(Life Technologies,目录号A21052)。
结果如图5所示,通过激光共聚焦检测流感病毒复制早期vRNP在细胞内的分布情况发现,在阴性对照DMSO处理组和AMG7703处理组随着病毒感染时间的延长,vRNP入核情况没有差异,说明AMG7703激活FFAR2后不影响vRNP入核,排除了其对病毒复制早期的影响。
五、AMG7703抑制高剂量H1N1流感病毒单轮感染时晚期病毒蛋白的表达
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述30、20μM AMG7703工作液于12孔板中,于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将H1N1(WSN)病毒以MOI=5的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述30、20μM AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的3、6和9h时弃掉细胞上清液,用1×loading裂解细胞,通过免疫印迹法进行检测在流感病毒单轮复制过程中各病毒蛋白的表达,进而确定AMG7703处理后影响病毒复制周期的具体阶段。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1、PA和NP抗体(本实验室制备鼠单克隆抗体)、HA抗体(Sino Biological,目录号11692-T54)、NA抗体(GeneTex,目录号GTX629696)、M1抗体(GeneTex,目录号GTX125928)、M2抗体(GeneTex,目录号GTX125951)、NS1抗体(GeneTex,目录号GTX125990)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight 800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图6所示,与阴性对照组相比,30和20μM AMG7703处理后均能够对不同病毒蛋白的表达产生抑制作用,但AMG7703处理后对晚期蛋白(HA、NA、M1和M2)的抑制作用明显得强于对早期病毒蛋白(PB2、PB1、PA和NP)的抑制作用;同时我们还发现尽管AMG7703的用药剂量降低了,但其对晚期蛋白(HA、NA、M1和M2)的抑制作用仍然很强,进一步地说明AMG7703激活FFAR2后主要是通过影响病毒生活周期的晚期阶段来抑制病毒复制。
六、利用CellTiter Glo试剂盒测定不同浓度的AMG7703对HEK293T细胞的毒性。
将HEK293T细胞悬液平铺于96孔板中,每孔中加入100μL含10%胎牛血清的DMEM培养基(购自Gibco,目录号C11995500BT),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的DMEM培养基分别稀释成50、40、30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入150μL上述不同浓度的AMG7703工作液(各三孔)于96孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在孵育24h后,每孔中加入100μL CellTiter-Glo reagent(购自Promega,目录号G7572)于37℃培养箱裂解10min,荧光数值用GloMax 96MicroplateLuminometer(Promega)进行检测。每个处理均设置3个重复孔,每个实验进行至少2次重复。实验结果利用Graphpad Prism 5数据处理软件进行作图,并进行统计分析。
结果如图7所示,与阴性对照组相比,50μM AMG7703能够对HEK293T细胞活力产生接近20%的影响,40μM AMG7703能够对HEK293T细胞活力产生接近10%的影响,30、20μMAMG7703对HEK293T细胞活力无影响,因此后续在HEK293T细胞中实验使用的工作浓度为30和20μM。**代表p<0.05。
七、不同浓度的AMG7703对三种聚合酶蛋白和NP蛋白的表达无影响
将HEK293T细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的DMEM培养基(购自Gibco,目录号C11995500BT),放置于37℃含5%CO2细胞培养箱中培养;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL Opti-MEM。分别将1μg pCAGGS WSNPB2、1μg pCAGGS WSN PB1、1μg pCAGGS WSN PA、0.5μgpCAGGS WSN NP蛋白表达质粒加入到Opti-MEM中混合均匀,每管中分别加入3μLP3000转染试剂混合均匀后,室温静置5min,然后每管中分别加入3μL LipofectamineTM3000转染试剂混合均匀后,室温静置15min后,缓慢滴加到12孔板中。100mM贮存液用Opti-MEM培养基分别稀释成30和20μM的工作液;在转染后的24h,每孔中分别加入1mL 30和20μM的工作液,放置于37℃含5%CO2细胞培养箱孵育12h,用1×loading裂解细胞,通过免疫印迹法进行检测AMG7703对三种聚合酶蛋白(PB2/PB1/PA)和NP蛋白的表达影响。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1、PA和NP抗体(本实验室制备鼠单克隆抗体)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图8所示,与阴性对照组相比,30和20μM AMG7703对三种聚合酶蛋白(PB2/PB1/PA)和NP蛋白的表达均无影响。
八、不同浓度的AMG7703抗甲流H1N1亚型流感病毒复制的作用
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成40、30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述不同浓度的AMG7703工作液(各三孔)于12孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将甲流H1N1(FZ09)病毒以MOI=0.1的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述不同浓度的AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的12和24h时采集细胞上清液,进行蚀斑滴定。在病毒感染后的24h时弃掉细胞上清液,用1×loading裂解细胞,通过免疫印迹法进行检测在流感病毒多轮复制过程中AMG7703对甲流H1N1病毒蛋白的表达影响,进而明确AMG7703对甲流H1N1病毒的抑制作用。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1和NP抗体(本实验室制备鼠单克隆抗体)、M1抗体(GeneTex,目录号GTX125928)、NS1抗体(GeneTex,目录号GTX125990)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight 800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图9中A所示,40μM AMG7703在12和24h分别能够抑制甲流H1N1(FZ09)病毒复制达10.2倍和18.4倍;30μM AMG7703在12和24h分别能够抑制甲流H1N1(FZ09)病毒复制达4.6倍和7.4倍;20μM AMG7703在12和24h分别能够抑制甲流H1N1(FZ09)病毒复制达2.7倍和4.3倍;Western blotting检测结果显示30、20μM AMG7703不仅能够抑制聚合酶相关蛋白(PB2、PB1和NP)的表达,还能够抑制晚期蛋白M1和NS1表达(图9中B)。
上述实验结果显示,AMG7703在激活FFAR2后能够抑制甲流H1N1病毒的复制。
九、不同浓度的AMG7703抗H5N1亚型流感病毒复制的作用
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成40、30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述不同浓度的AMG7703工作液(各三孔)于12孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将H5N1(AH05)病毒以MOI=0.1的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述不同浓度的AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的12和24h时采集细胞上清液,进行蚀斑滴定。在病毒感染后的24h时弃掉细胞上清液,用1×loading裂解细胞,通过免疫印迹法进行检测在流感病毒多轮复制过程中AMG7703对H5N1(AH05)病毒蛋白的表达影响,进而明确AMG7703对H5N1(AH05)病毒的抑制作用。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1和NP抗体(本实验室制备鼠单克隆抗体)、M1抗体(GeneTex,目录号GTX125928)、NS1抗体(GeneTex,目录号GTX125990)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight 800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图10中A所示,40μM AMG7703在12和24h分别能够抑制H5N1(AH05)病毒复制达6.9倍和4.2倍;30μM AMG7703在12和24h分别能够抑制H5N1(AH05)病毒复制达3.4倍和3.7倍;20μM AMG7703在12和24h分别能够抑制H5N1(AH05)病毒复制达1.8倍和2.5倍。同时,Western blotting结果显示30、20μM AMG7703对H5N1(AH05)病毒晚期蛋白M1表达产生明显的抑制作用(图10中B),进一步夯实了AMG7703对高致病性H5N1流感病毒复制的抑制作用。
上述实验结果显示,AMG7703在激活FFAR2后能够抑制H5N1病毒的复制。
十、不同浓度的AMG7703抗禽源H9N2亚型流感病毒复制的作用
将A549细胞悬液平铺于12孔板中,每孔中加入1mL含10%胎牛血清的F-12K培养基(购自Gibco,目录号21127030),放置于37℃含5%CO2细胞培养箱中培养;100mM贮存液用含0.3%BSA的F-12K培养基分别稀释成40、30、20μM的工作液;当细胞密度达95%时,弃掉细胞上清液,用PBS洗涤一次,加入1mL上述不同浓度的AMG7703工作液(各三孔)于12孔板中,放置于37℃含5%CO2细胞培养箱中,同时设立DMSO(购自Sigma,目录号D2650)处理组作为阴性对照。在药物预处理3h后,将禽H9N2(SC197)病毒以MOI=0.1的剂量感染A549细胞,在37℃含5%CO2细胞培养箱中孵育1h后,用PBS洗涤两次,加入1mL上述不同浓度的AMG7703工作液后放置于37℃含5%CO2细胞培养箱中培养,在病毒感染后的12和24h时采集细胞上清液,进行蚀斑滴定。在病毒感染后的24h时弃掉细胞上清液,用1×loading裂解细胞,通过免疫印迹法进行检测在流感病毒多轮复制过程中AMG7703对禽源H9N2(SC197)病毒蛋白的表达影响,进而明确AMG7703对禽源H9N2(SC197)病毒的抑制作用。
免疫印迹实验中使用PB2抗体(GeneTex,目录号GTX125926)、PB1和NP抗体(本实验室制备鼠单克隆抗体)、M1抗体(GeneTex,目录号GTX125928)、NS1抗体(GeneTex,目录号GTX125990)、GAPDH抗体(Proteintech,目录号60004-1-Ig)、DyLight 800goat anti-rabbit IgG(H+L)(Immunoway,目录号RS23920)和DyLight 680goat anti-mouse IgG(H+L)(Immunoway,目录号RS23710)。
结果如图11中A所示,40μM AMG7703在12和24h分别能够抑制H9N2(SC197)病毒复制达29.9倍和55.4倍;30μM AMG7703在12和24h分别能够抑制H9N2(SC197)病毒复制达25.2倍和40.1倍;20μM AMG7703在12和24h分别能够抑制H9N2(SC197)病毒复制达16.2倍和15.9倍。同时,Western blotting结果显示30、20μM AMG7703对聚合酶相关蛋白(PB2、PB1和NP)和晚期蛋白M1和NS1表达产生明显地抑制作用(图11中B),进一步夯实了AMG7703对禽源H9N2流感病毒复制的抑制作用。
上述实验结果显示,AMG7703在激活FFAR2后能够抑制禽源H9N2病毒的复制。
Claims (1)
1.AMG7703或其药学上可接受的盐在制备抗流感病毒感染的药物中的用途,所述AMG7703的结构如下所示:所述流感病毒为H1N1、H5N1或H9N2亚型流感病毒。
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