靶向血凝素蛋白的高致病性禽流感H5N1进入抑制剂的研究
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摘要
研究背景：禽流感自被发现以来,一直对人类健康和社会经济发展构成了严重的威胁,每年全球超过15%人口会感染流感,导致超过50万人死亡。历史上一些著名的禽流感疫情,例如1918年的西班牙流感造成数千万人死亡。最近2009年爆发了新型的猪流感H1N1在213个国家中造成数百万人感染。其中最令人担心的是高治病性禽流感H5N1,1997年在香港发现人类也会感染H5N1后,引起人们高度关注。在2003年,在中国和东南亚出现了人感染H5N1,接着疫情迅速向全球扩散至东欧和非洲。随后,H5N1疫情一直有零星爆发,2011年在香港和西藏拉萨都出现了H5N1疫情。到目前为止,全球累计596例人感染H5N1,死亡350例,死亡率高达60%。虽然目前H5N1的传染途径主要是禽传人,由于禽流感病毒在宿主的复制过程中会通过突变获得宿主唾液酸受体的识别特异性,因此人们非常担心H5N1通过高频突变适应了以人类为宿主,获得人传染人的能力,造成严重疫情。
目前获批有效的抗流感药物有两类,M2离子通道阻滞剂和神经氨酸酶抑制剂。前者的缺点是短时间内可出现耐药,并且对B型流感病毒无效。自从2003年后,由于全球流行的甲型禽流感都对金刚烷胺和金刚烷乙胺耐药,故此类药物在临床上已很少使用。后者价格昂贵,但对A、B型流感病毒均有效,不良反应小。但是目前流行的甲型流感毒株对奥司他韦均出现一定程度的耐药性。
最近的研究使人们把目光关注在病毒进入抑制剂上。病毒进入抑制剂也称为融合抑制剂,作为一种新型的抗病毒药物,最早应用于治疗HIV。此类药物,可以是小分子化合物、抗体、多肽,作用于病毒进入细胞的不同环节,通过竞争结合病毒表面蛋白或细胞受体,干扰了这些蛋白的正常功能,阻止病毒进入靶细胞,起到抵抗病毒感染的作用。禽流感病毒的包膜蛋白血凝素介导与宿主细胞唾液酸受体的结合,病毒的进入和融合过程,是一个潜在的进入抑制剂靶点。
近年研究发现TBHQ、BMY-27709、CL-61917、CL-385319等小分子化合物能够阻止酸性环境引起的HA结构重组,起到抗病毒的作用。TBHQ对H3亚型流感病毒有抑制活性,对H5亚型无效。对TBHQ与血凝素的共结晶解析发现TBHQ结合于HA2亚基上的Arg54、Glu57、Glu97残基,稳定了三聚体,使HA在酸性条件下保持中性状态的结构,阻止了病毒的进入。N-取代哌啶化合物CL-61917和CL-385319对H1和H2亚型禽流感有抑制活性,IC50达到μM水平。分析化合物的耐药病毒株发现HA蛋白序列有氨基酸突变,这个氨基酸位于HA三聚体茎部区域靠近HA2融合肽的部位。BMY-27709,能够抑制A/WSN/33病毒和H1亚型和H2亚型的复制,IC50在3-8μM。对BMY-27709耐药的毒株HA基因序列分析发现了耐药突变位点位于HA2亚基的N端,表明化合物是结合于HA2亚基N端来阻止血凝素介导的膜融合。Stachyflin能够有效地阻止H1亚型和H2亚型禽流感的感染,IC50达到μM水平。对Stachyflin耐药的毒株HA基因序列分析发现了两个点突变,K51R、K121E,位于HA2亚基。CR6261单抗是新近发现的广泛中和抗体,对多种亚型的流感病毒均有效,在动物实验中能保护小鼠抵抗致死量病毒的攻击。晶体结构显示,它们都是结合于HA2上的茎部区域,使HA在酸性条件下保持中性状态的结构,阻止了病毒的进入。
以上研究表明血凝素的HA2亚基是一个潜在的小分子药物靶点和疫苗靶点。但是上述的有抑制流感病毒活性的化合物只限于特定的亚型,目前还没有对高治病性禽流感H5N1有抑制活性的报道。并且上述化合物的耐药株的突变位点都位于HA2亚基的茎部区域。这个区域分布着大量的可电离氨基酸,在病毒的进入过程中起着重要的作用。突变研究发现其中一些氨基酸残基会影响HA三聚体的稳定性,使融合PH升高。目前只获得了TBHQ与HA的共结晶,研究清楚了其抗病毒的机制。但其他化合物与血凝素结合的模式,抑制病毒进入的机制还不清楚。
目的和意义：在早前的研究中,我们发现了CL-385319对H5N1A/Vietnam/1194/2004有抑制作用,并且初步确认了靶点是血凝素的HA2亚基。本研究中旨在建立多种具有代表性毒株的高致病性禽流感H5N1假病毒,用于进一步确认CL-385319的抗H5N1作用和筛选其他小分子化合物；利用点突变技术往HA上引入突变位点,观察突变位点和CL-385319抑制活性的关系,确认CL-385319在血凝素上的作用位点；深入研究CL-385319与血凝素结合位点的结合模式,探讨CL-385319阻止流感病毒进入的机制；并以CL-385319为先导化合物进行结构改造得到一系列衍生物,评价衍生物的抗H5N1禽流感活性,寻找活性更好的化合物,确认衍生物的作用位点,初步总结构效关系,为进一步的药物结构优化和改造提供了空间。
方法和结果：实验数据均采用Mean±SD表示,采用SPSS13.0软件进行数据分析。先进行方差齐性检验,方差齐时采用one-way ANOVA方法进行显著性检验统计分析,多重比较采用LSD方法分析；方差不齐时采用近似F检验的Welch方法进行显著性检验统计分析,多重比较采用Dunnetf’s T3进行分析。p<0.05为差异有统计学意义。
作为先导化合物必须要有广谱的抗病毒活性,首先我们要建立多种具有代表性毒株的高致病性禽流感H5N1假病毒。我们将表达HA和NA的质粒,与HIV包膜蛋白基因缺失的骨架质粒pNL4-3.luc.R-E'共同转染293T细胞,收获上清,得到具有单轮感染活性的假病毒颗粒。本文一共构建了5种人源H5N1假病毒,分别是A/Qinghai/59/2005, A/Xinjiang/1/2006、A/Anhui/1/2005、A/HongKong/156/1997及A/Thailand/Kan353/2004。这5株H5N1假病毒都能成功感染MDCK细胞。在不同的药物浓度下,我们检测了CL-385319对5株H5N1假病毒的抑制活性。结果表明,CL-385319对5种H5N1假病毒均有抑制作用,并特异性作用于病毒的进入环节。对A/Qinghai/59/2005、A/Xinjiang/1/2006, A/Anhui/1/2005、A/Hong Kong/156/1997及A/Thailand/Kan353/2004毒株的IC50分别是1.5±0.13μM、2.22±0.24μM、0.37±0.12μM和2.62±0.08μM.这表明CL-385319具有广谱的抗H5N1禽流感活性。我们对这5株毒株的HA蛋白序列进行了对比,发现HA1段差别较大,HA2段同源性高,这符合我们早前的实验结果,CL-385319的作用位点在HA2亚基上。
要阐明CL-385319的作用机制,必须要明确化合物的具体作用位点。前人发现CL-385319有抑制H1亚型流感的活性,并且发现了HA2的两个突变N502D, F1102S能让H1亚型流感毒株产生耐药性。根据HA的同源性分型,H1,H5亚型流感属于同一组别,H3亚型属于另一组别。通过序列比对,我们发现H1N1与H5N1的HA2序列同源性达到78.5%,并且H5N1的HA2同样具有N502和F1102两个残基。利用SYBYL7.3软件的子程序Surflex-dock在H5N1血凝素的F1102残基处发现了一个空腔,位于血凝素的茎部区域。这个空腔是由Gl2、L22、K51、E1052、R106、T1072、L1082、D1092、F1102、T221、M241和E251氨基酸残基构成。
为了明确CL-385319在HA上的具体作用位点,我们以A/Qinghai/59/2005-HA质粒为模板,引入单点突变位点：包括M241A、E251A、 R3221A、K432A、D462A、G472A、V482A、N502D、K512A、E1052A、R1062A、 T1072A、pD1092A和F1102S。将鉴定为含有目的突变基因的阳性克隆质粒和NA质粒,与HIV包膜蛋白基因缺失的骨架质粒pNL4-3.luc.R-E共同转染293T细胞包装出假病毒颗粒。我们测定了14个突变位点假病毒感染MDCK细胞的荧光发光值。Levene方差齐性分析检验结果是,F=4.039,P<0.000,方差不齐(P<0.05)；采用近似F检验的Welch方法,F=3460.069,P<0.000,采用Dunnett'sT3检验各种点突变与野生型(WT)的差异,发现G472、K512A、R1062A、D1092A与WT没有显著性差异,P值分别是(0.951,0.857,1.000,1.000),其余突变假病毒都与野生型有显著性差异,P值均<0.000。而且带有N502D、E1052A、T1072A点突变的假病毒与Blank没有显著性差异,P值均大于0.05,这表明假病毒完全丧失感染能力；而F1102S假病毒感染性较低,与野生型假病毒有显著性差异,P<0.000,这表明HA2的N502、E1052、T1072、F1102对病毒的进入起着关键的作用。接着在不同的浓度下,我们检测了突变前后的假病毒对CL-385319的敏感性。结果发现,V482A、M241A、F1102A这三个点突变假病毒均对CL-385319耐药。E251A、D1092A突变的假病毒敏感性增强。其他位点的突变假病毒对CL-385319敏感性变化均不明显。为了进一步确认假病毒感染力丧失／降低以及耐药性是由氨基酸突变引起的,我们用ELISA检测了突变假病毒裂解液中P24的含量,Levene方差齐性分析检验结果是F=1.545,P=0.155,方差齐性,采用one-way ANOVA方法,F=0.633,P=0.816,突变组与野生型组没有显著性差异,这表明突变假病毒与野生型的P24抗原含量在同一个水平,所有带有突变的假病毒均包装成功。我们还用Western blot检测突变H5N1假病毒的血凝素表达量,发现N502D、E1052A、T1072A、V482A、M241A、F1102A的突变假病毒的血凝素都正常表达。这些结果表明CL-385319在HA上的作用位点是在HA的茎部区域,V482A、M241A、F1102A是CL-385319直接结合的氨基酸残基。我们进一步地分析了这些关键氨基酸在人源H5N1禽流感中的保守性,对目前已知的405列HA序列进行比对发现V482、M241、F1102、E1052和T1072都是高度保守的。
为了阐明CL-385319与血凝素结合位点的结合模式,探讨CL-385319阻止流感病毒进入的机制,我们利用分子对接和分子动力学模拟的方法进行了研究。我们首先用Autodock对接软件得到了CL-385319与HA三聚体对接复合物的初始结构,接着运用Amber软件进行了长达47ns动力学模拟,用MM GBSA计算了体系的结合自由能,用丙氨酸扫描方法预测了与CL-385319作用的关键残基,用量化计算软件Laplacian计算了CL-385319与靶穴残基间氢键的相互作用。我们对动力学模拟中能量最低点的构象进行了分析。CL-385319结合位点在HA三聚体的茎部区域,由相邻的两个HA单体构成MM_GBSA结果显示复合物的结合自由能是-40.6109kcal mol-1,这表明结合过程是自发进行的.CL-385319通过π-π相互作用嵌合在HA2的F1102和HA1的M241氨基酸残基之间,形成一个三明治的结构,并且与M241、G12、R1062、T1072形成氢键。π-π相互作用和氢键作用对CL-385319与HA形成稳定的复合物起了主要作用。
我们比对了动力学模拟过程中HA的平均结构和HA的晶体结构(pdb:2IBX),发现HA的晶体结构中原本不存在结合的靶穴,在初始结构中,F1102和M241之间的距离不足以让CL-385319的苯环部分插进去。而在动力学模拟过程的平均结构中,我们发现靶穴的氨基酸残基位置的移动都在RMSD的范围内除了M241的位置发生了明显变化,M241残基朝着F1102相反的方向往外移动,使F1102和M241之间的距离增加到8.001A,产生了一个可供CL-385319的苯环部分结合的空腔,最终形成三明治结构。在分析MD过程中的结构时还发现V482残基对CL-385319与HA结合起着关键的作用。在MD的早期构象中,CL-385319的三氟甲基与V482形成了三个氢键,起到稳定的作用,而此时F1102和M241之间的距离还没有增加。随着MD过程的进行,三氟甲基与V482的氢键断裂,F1102和M241的距离增加,与CL-385319的苯环形成三明治结构,体系结合自由能降低。这些计算结果表明CL-385319在与HA的结合中,HA结合位点的氨基酸残基位置发生了变化来与CL-385319结合,这符合Koshland提出的诱导契合学说。
CL-385319的关键结合位点位于血凝素的茎部区域,血凝素只有这个区域的可电离氨基酸会因水解时融合肽的插入而被深埋入三聚体的内部,两者之间的氢键作用和盐桥作用在中性状态下能稳定HA1/HA2的结构。在酸性条件下,氢离子增加,这些可电离氨基酸的质子化状态改变,使HA进入融合态结构。前人的突变研究也显示这些残基与低pH触发的HA结构重组直接相关。E1052K突变能增加血凝素在低pH下的稳定性,而R1062H突变增强了H2亚型血凝素在低pH下的稳定性,并且阻止了HA发生融合结构重组。然而这个区域的氨基酸残基从来没有明确地与病毒进入抑制剂联系起来,因此弄清楚CL-385319稳定血凝素的机制会对新的进入抑制剂设计带来启发。E1032,E1052,R1062和T1072位于HA茎部区域。E1052A和T1072A单点突变使H5N1假病毒丧失了感染能力。分子动力学模拟显示CL-385319与R1062和T1072发生氢键相互作用,稳定了这个节段的构象,可能改变了这些残基的质子化状态,阻止了酸性破坏残基间的电荷作用。研究报道融合肽的G12E突变能够使膜融合过程不能进行,CL-385319能与G12形成氢键,对融合肽起到稳定的作用,阻止其移动。另外,HA1与HA2亚基的分离是HA结构重组的第一步。我们的动力学模拟结果表明CL-385319的苯环与HA1亚基上的M24和HA2亚基上的F110由于π-π作用形成三明治结构,起到固定HA1和HA2亚基的作用。综上所述,在融合pH时,CL-385319通过稳定HA的中性结构,阻止了病毒进入过程中所必需的结构重组,起到抗病毒的作用。
我们以CL-385319为先导化合物进行结构修饰,合成了一系列衍生物。我们利用假病毒体系评价了衍生物抗H5N1的活性,一共筛选出26个活性化合物,其中有4个活性化合物达到nM水平,活性最好的化合物是3-氟-5-三氟甲基-N-((2-噻吩基)乙基)苯甲酰胺。为了确定新化合物的作用位点,我们检测了这26个衍生物对V482A、F1102S和M241A这三种H5N1突变体假病毒的敏感性。结果显示这26个衍生物对这三株假病毒都没有抑制活性,这表明衍生物是作用于CL-385319一样的作用位点,也就是血凝素的茎部区域。其抗病毒的机理也是稳定HA的中性结构,阻止了酸性低pH触发的血凝素结构重组,从而阻止膜融合和病毒进入。我们测定了活性较好的衍生物A11、A12、A13、A14对神经氨酸酶的抑制活性,Levene方差齐性分析检验结果是F=2.123,P=0.115,采用One-way ANOVA方法,F=960.989,P=0.000,有显著性差异,用LSD进行多重比较,分析化合物组与No compound组的差异,发现A1l、A12、A13、CL-385319与No compound组没有显著性差异,P值均大于0.05,这表明A11、A12、A13、A14化合物对神经氨酸酶没有抑制活性。
我们初步总结了26个衍生物的构效关系。我们发现衍生物的立体构型与CL-385319越接近的,活性越好。我们发现化合物母核上的取代基与活性有明确的关系。针对R4基团,F取代CF3或CF3取代H都能增强抗流感活性。T基团的空间位阻越大活性越差.空间位阻可能影响了化合物与R1062,T1072和M241残基形成氢键作用。化合物的苯甲酰胺的氧原子与G1残基形成氢键,对活性很关键,把甲酰胺取代成-SO2NH2和-COO-,化合物的活性则明显下降。
结论
1.CL-385319具有广谱的抗H5N1禽流感活性,对A/Qinghai/59/2005、 A/Xinjiang/1/2006、A/Anhui/1/2005、A/HongKong/156/1997和A/Thailand/Kan353/2004都有很强的抑制作用。
2.CL-385319的作用位点位于H5N1的HA的茎部区域的保守空腔,由G12、L22、K512、E1052、R1062、T1072、L1082、D1092、F1102、T221、 M24和E251氨基酸残基构成,其中V482A、M241A、F1102A是CL-385319直接结合的氨基酸残基。
3.CL-385319与HA的结合是一个诱导契合的过程。
4.CL-385319与HA的结合,稳定了HA的中性结构,阻止了pH触发的融合结构重组,阻止了禽流感病毒的进入。
5.以CL-385319为先导化合物合成的一系列衍生物具有抗H5N1和H1N1禽流感病毒活性,验证了药物靶点的真实性,为进一步化合物结构优化和改造提供了空间。
Background:Influenza continues to pose serious threats to public health worldwide since seasonal influenza epidemics can affect up to15%of the population and result in more than500,000deaths worldwide each year, while some influenza pandemics, like the1918flu pandemic (the Spanish Flu), may lead to millions of deaths. In2009, a new influenza pandemic caused by a novel swine-origin influenza A virus (S-OIV) H1N1has resulted in millions of infections in more than213countries. Highly pathogenic avian influenza (HPAI) H5N1viruses have caused350fatal cases among a total of596infected individuals, with a case-fatality rate of59%. The HPAI H5N1virus emerged as a human pathogen in1997within the Hong Kong. Since late2003, the H5N1virus has expanded its geographical range to affect poultry in East and Southeast Asia. Subsequently, H5N1virus has spread to wild birds across Eurasia and as far west as England and Africa. Since viruses are passaged in a particular host they can adapt to that host by mutating the receptor-binding site in the viral HA, there is a growing concern about H5N1acquiring human-to-human transmissibility.
At the current time, only2classes of influenza antiviral agents are currently licensed M2ion channel inhibitors and neuraminidase inhibitors. The M2ion channel inhibitors (amantadine and rimantadine) are ineffective against influenza B strains and, influenza A virus resistant to amantadine and rimantadine can emerge quickly during treatment. Since2003, the M2ion channel inhibitors are recognized to have extremely limited clinical utility because ofworldwide influenza A virus resistance to these2drugs. Neuraminidase inhibitors are both effective against influenza A and B strains. But clinical virus resistant to oseltamivir has been reported.
Entry inhibitors, also known as fusion inhibitors, are a class of antiretroviral drugs, used in combination therapy for the treatment of HIV infection. This class of drugs interferes with the binding, fusion and entry of virus to host cells. HA plays crucial roles in the early stage of influenza virus infection, including virus binding to host receptors, viral entry, and membrane fusion. It is a potential target for entry inhibitors.
Several small molecules that can block the pH-depended irreversible conformational changes of HA, such as TBHQ, BMY-27709, CL-61917, CL-385319Stachyflinwere shown to inhibit IAV infection. TBHQ can inhibit H3-subtype influenza virus, but not H5-subtype. TBHQ could induce drug-resistance mutations in HA2subunit, suggesting that it acts on the HA protein. X-ray crystallographic study indicates that TBHQ binds with HA2and locks the HA2trimer at the neutral-pH conformation, through the interaction of TBNQ with three ionizable amino acids in this site:Arg-54, Glu-57and Glu-97from HA2subnuit. CL-61917and CL-385319show inhibitory activity against H1and H2subtypes of IAVs with IC50at low micromolar levels. Analysis of HA genes of the mutant viruses resistant to these compounds showed that amino acid mutations clustered in the stem region of the HA. trimer in and near the HA2FP. BMY-27709can inhibit H1and H2subtype IAV infection through its specific interaction with the HA protein to repress its conformational change of HA induced by low-pH. Sequence analyses of the HA gene of the variants resistant to BMY-27709mapped the amino acidsubstitutions responsible for drug resistance to a region located near the N terminus of HA2. Stachyfl in is effective in inhibiting infection by H1and H2subtype IAVs with IC50at low μM level. It was shown that Stachy fl in interfered with low pH-induced conformational change of HA, thereby blocking HA-mediated virus-cell fusion. Analysis of the HA gene sequences of the IAV variants with resistance to Stachy fl in revealed that two amino acid substitutions, K51R and K121E, in the HA2subunit of the HA protein. Recent studies have indicated that monoclonal antibody(mAb) CR6261, which recognizes a highly conserved helical region in the membrane-proximal stem of HA1and HA2, could neutralize the virus by blocking conformational rearrangements associated with viral membrane fusion. However, until now, these ionic residues had not been correlated with properties of influenza fusion inhibitors. Until now, only the TBHQ antiviral mechanism has been resolved by X-ray crystallography. The mechanism of action of other entry inhibitors has not been uncovered yet.
These studies demonstrated that HA2subnuit, especially the stem region, to be an antiviral target for entry inhibiots and for inducing broad neutralizing antibody responses. But the molecules mentioned above just showed subtype-dependent activities, and small-molecule inhibitors which inhibit HPAI H5N1have not been reported so far. In addition, Ionic residues at or near the stem region are suggested to play a critical role in fusion activation because of the apparent relationship between their chemical environment and HA sensitivity to low pH. Mutational analysis showed that amino acids in this cavity are associated with low pH-induced conformational change.
Objection:We previously found that CL-385319can inhibit A/Vietnam/1194/2004H5N1in vitro and might target on HA2subunit. The present study aims to establish different isolate H5N1pseudovirus systems to confirm CL-385319antiviral activity and to screen other small-molecule compounds. To identify the critical binding site of CL-385319on HA by detailed site-directed mutagenesis. To uncover the molecular mechanism underlying the action of CL-385319by extensive computational simulations, this could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza. A series of derivatives which focused on structural variation of CL-385319were synthesized and evaluated their anti H5N1activity. The structure-activity relationship is obtained for further research.
Methods and Results:The data are presented as means±standard deviations. Levene's test for homogeneity of variance is performed. When the assumption of equal variances holds, One-Way ANOVA is preferable to the F statistic and LSD is performed to do pairwise multiple comparisons. When the assumption of equal variances does not hold, Welch is preferable to the F statistic and Dunnett's T3is performed to do pairwise multiple comparisons.
To test whether CL-385319may inhibit different isolate of H5N1IAV, we generated five H5N1pseudoviruses. The plasmids encoding HA of different isolate H5N1IAV, including A/Thailand/Kan353/2004, A/Qinghai/59/2005, A/Xinjiang/1/2006, A/Anhui/1/2005, A/Hong Kong/156/1997, as well as the A/Thailand/Kan353/2004Nl subtype NA plasmid, were cotransfected with an HIV-1env deleted backbone plasmid pNL4-3.1uc.R-E-in293T cells. We harvested H5N1pseudovirus particles from the culture supernatants. These five H5N1pseudoviruses were able to infect MDCK and maintain significant high infectivity. CL-385319could inhibit these five H5N1pseudoviruses with IC50of4.00±0.38μM, 1.50±0.13μM,2.22±0.24μM,0.37±0.12μM,2.62±0.08μM, respectively. However, CL-385319had no effect on VSV-G pseudovirus infection, demonstrating that the compound specifically interferes with the entry of influenza virus. We compare the HA sequences of these five H5NA isolate and found that the amino acid sequence identity of HA1is releatively low, that of HA2is much higher. This is consistent with our previous study, the binding sites of CL-385319located in HA2.
It is needed to identify the critical residues for CL-385319binding first to uncover the detailed binding mechanism. Previous study showed that CL-385319is effective in inhibiting infection by HI subtype IAVs, but N502D, F1102S rendered the virus highly resistant to CL-385319. Based on phylogenetic analysis, H1and H5belong to Group1, while H3belongs to Group2of IAV HAs (Russell et al.,2008). The amino acid sequence identity of HA2subunit between H5and H1is81%. Specially, the critical residues for the activity of CL-385319, N502and F1102, identified through CL-385319-resistant H1N1virus, are identical between H5and H1or H2subtypes. Using Surlex-Dock program of SYBYL7.3, a cavity was found in the stem region of the HA near the fusion peptide. The cavity is surrounded by a few residues:G12, L22, K512, E1052, R1062, T1072, L1082, D1092, F1102, T22,, M241, E251and R3221.(1refers to HA1subunit,2refers to HA2subunit)
We identified the critical residues for CL-385319binding on H5N1hemagglutinin using site-directed mutagenesis analysis. We generated a series of mutant HA pseudoviruses based on A/Qinghai/59/2005-HA plasmid. The residues surrounded the cavity including:M24h E251, R3221, K432, D462, G472, V482, N502, N502, K512, E1052, R1062, T1072, D1092, and F1102were selected and substituted by alanine. The mutant pseudovirus susceptibility to CL-385319was tested. The result of Levene's test for homogeneity of variance is F=4.039, P<0.000. Welch is preferable to the F statistic, F=3460.069, P<0.000; Dunnett's T3is performed to do pairwise multiple comparisons. Results showed that the RLU of G472A, K512A, R1062A, D1092A mutations have no significance with that of WT, P=0.951,0.857,1.000,1.000, respectively. The rest of mutations have significance with WT, P<0.000. Results showed that the M241A, F1102S and V482A mutations rendered the virus highly resistant to CL-385319, suggesting that M241, F1102and V482are the critical residues involved in the binding of CL-385319. M241, F1102and V482are highly conserved in all H5N1strains. However, D1092A and E251A mutations resulted in increased sensitivity of pseudoviruses to CL-385319. One possible reason is that the mutations of E251A and D1092A cause changes of conformation and hydrophobicity of the cavity, which might increase the susceptibility of F1102and M241.to CL-385319. Interestingly, the individual N502D, E1052A and T1072A mutation eliminated pseudovirus infection, P>0.05vs Blank; F110S showed moderate infectivity, P=0.000vs WT, while Western blotting analysis showed that none mutation affected HA protein expression on the surface of pseudovirus. The amount of p24contained in the pseudovirus tested by HIV-1p24ELISA were on the same level. The result of Levene's test for homogeneity of variance is F=1.545, P=0.155. One-way ANOVA is preferable to the F statistic, F=0.633, P=0.816. There is no significance between mutation group and Wildtype. These results suggest that N502, E1052, T1072and F1102are critical for viral entry and that these mutations might interfere with the structural rearrangement needed for membrane fusion.
Furthermore, extensive computational simulations, including molecular docking, molecular dynamics (MD) simulations, MM_GBSA calculations, as well as charge density and its Laplacian calculations, have been carried out to uncover the detailed mechanism of CL-385319binding to HA. The CL-385319binding site is formed by HAl and HA2in one monomer. There are three identical binding sites per HAtrimer. The calculated binding free energy of the complex was-40.6109kcal mol-1, which indicated that the binding of CL-385319to HA could increase the stability of HA protein. Consistent with the experimental mutagenesis result, the π-π interactions between CL-385319and residues F1102and M241make important contributions to the binding affinity. In addition, CL-385319can form hydrogen bonds with the residues of E1052, R1062and T1072.
As suggested by Koshland, the induced-fit model describes the capability of a proper ligand to induce specific structural change of the active site such that the protein is able to achieve the final key-lock state.We found that the binding of CL-385319to HA is a process of "induced fit". In the CL-385319-HA complex structure derived from molecular docking, which was used as starting structure for molecular dynamics simulation, the trifluorophenyl group of CL-385319formed three important H-bonds interaction with V482-The averaged structure of47ns MD simulation shows that the benzene ring of CL-385319was sandwiched by F1102and M24] via π-π interactions, while the H-bonds interaction between trifluorophenyl group and V482was not observed in the lowest energy structure. Therefore, we believe that the H-bond interaction between the trifluorophenyl group and V482stabilizes the inhibitor in the early stage of MD and facilitates the formation of π-π interactions. Originally, no binding pocket was observed between F1102and V482. However, during MD simulation, a significant conformational change of the M24] residue was observed, as it shifted away from F1102to accommodate CL-385319. In addition, the free energy of CL-385319binding to HA calculated by the MM_GBSA method showed that the binding process is thermodynamically favorable. Therefore, we conclude that CL-385319binding to HA occurs through the induced-fit pathway.
The critical residues for CL-385319binding locate in the stem region of HA. Ionic residues at or near this region are the only residues that experience different solvent environments before and after the insertion of fusion peptide into the cavity at the trimer interface, a necessary priming step of HA fusion. These residues are therefore suggested to play a critical role in fusion activation because of the apparent relationship between their chemical environment and HA sensitivity to low pH. Mutational analysis showed that amino acids in this cavity are associated with low pH-induced conformational change. For example, the E1052K mutation increased the stability of HA protein in low-pH, while the R1062H mutation improved the stability of H2-typed HA and inhibited the irreversible conformational rearrangement at fusogenic pH14. However, until now, these ionic residues had not been correlated with properties of influenza fusion inhibitors. Therefore, understanding how CL-385319stabilizes HA could provide a clue for the development of new influenza fusion inhibitors. In H5N1-typed HA2, E1032, E1052, R1062and T1072are located in the stem region which experienced different environments as described above. Mutagenesis results showed that E1052A and T1072A mutation eliminated pseudovirus infectivity. Molecular dynamics simulation showed that CL-385319has hydrogen-bonding interactions with residues of R1062and T1072. These interactions might interfere with the disruptions of inter-subunit ionic interactions induced by low-pH. Meanwhile, CL-385319has hydrogen-bonding interaction with residues of G12, the first residue of HA fusion peptide. It is known that G12E mutation abolish HA-mediated mutation. CL-385319might stabilize the conformation of the fusion peptide through this interaction. In addition, the dissociation of HA1from HA2is the first step for HA conformational rearrangement. Previous study showed that CL-385319can inhibit the proteolysis of purified HI-typed HA in low-pH. We demonstrated that the π-π interaction between CL-385319and the residue of F1102and M24] may provide a cross-linking of the HA trimer, thus inhibiting the dissociation of HA1and HA2; even the HAO was proteolyzed into HA1and HA2. As a consequence, CL-385319binding stabilizes the neutral pH conformation of HA, rendering its membrane fusion inactive.
A series of derivatives which focused on structural variation of CL-385319were synthesized and evaluated for antiviral activities against H5N1influenza virus.26analogues showed antiviral activities, four of them were found to be highly potent with IC50at nM level. The most effective compound was3-fluoro-5-(trifuoromethyl)-N-(2-(thiophen-2-yl)ethyl)benzamide. All of the V482A, F1102A and M241A mutant H5N1were resistant to these analogues, which suggested that they bind the same side and adopt the same mechanism to against H5N1as CL-385319did. All, A12, A13, A14were tested for the activity of N1-typed neuraminidase. The result of Levene's test for homogeneity of variance is F=2.123, P=0.115. One-way ANOVA is preferable to the F statistic, F=960.989, P<0.000; LSD is performed to do pairwise multiple comparisons. There is no significance between compound group and No compound group, P>0.05. The result showed that A11,A12, A13, A14have no activity on N1-typed neuraminidase
We further analysed the structure-activity relationship. Those analogues which have similar steric structure with CL-385319show higher antiviral activities. We found that replacement of R4-CF3-with F-group or H-with CF3-enhances the inhibitory activity. The steric hindrance of T-receptor affect activities significantly, analogues with higher antiviral activities have less steric hindrance. This might be associated with the formation of hydrogen bonds between compounds and R1062, T1072and M241residues.
Conclusion
1. CL-385319has potent inhibitory activity against a broad range of H5N1sublineages, including A/Thailand/Kan353/2004, A/Qinghai/59/2005, A/Xinjiang/1/2006, A/Anhui/1/2005and A/Hong Kong/156/1997.
2. The binding cavity of CL-385319locates in the stem region of HA and, is surrounded by a few residues:G12, L22, K512, E1052, R1062, T1072, L1082, D1092, F1102, T22,, M241, E251and R3221. The critical residues for CL-385319binding are F1102, M24, and V482.
3. CL-385319binding to HA occurs through the induced-fit pathway.
4. CL-385319binding stabilizes the neutral pH conformation of HA, rendering its membrane fusion inactive.
5. CL-385319derivates have potential inhibitory activity against H5N1and H1N1influenza virus. This confirmed the existance of binding pocket and provided a valuable foundation for the structure-based design of more potent influenza fusion inhibitors
目前获批有效的抗流感药物有两类,M2离子通道阻滞剂和神经氨酸酶抑制剂。前者的缺点是短时间内可出现耐药,并且对B型流感病毒无效。自从2003年后,由于全球流行的甲型禽流感都对金刚烷胺和金刚烷乙胺耐药,故此类药物在临床上已很少使用。后者价格昂贵,但对A、B型流感病毒均有效,不良反应小。但是目前流行的甲型流感毒株对奥司他韦均出现一定程度的耐药性。
最近的研究使人们把目光关注在病毒进入抑制剂上。病毒进入抑制剂也称为融合抑制剂,作为一种新型的抗病毒药物,最早应用于治疗HIV。此类药物,可以是小分子化合物、抗体、多肽,作用于病毒进入细胞的不同环节,通过竞争结合病毒表面蛋白或细胞受体,干扰了这些蛋白的正常功能,阻止病毒进入靶细胞,起到抵抗病毒感染的作用。禽流感病毒的包膜蛋白血凝素介导与宿主细胞唾液酸受体的结合,病毒的进入和融合过程,是一个潜在的进入抑制剂靶点。
近年研究发现TBHQ、BMY-27709、CL-61917、CL-385319等小分子化合物能够阻止酸性环境引起的HA结构重组,起到抗病毒的作用。TBHQ对H3亚型流感病毒有抑制活性,对H5亚型无效。对TBHQ与血凝素的共结晶解析发现TBHQ结合于HA2亚基上的Arg54、Glu57、Glu97残基,稳定了三聚体,使HA在酸性条件下保持中性状态的结构,阻止了病毒的进入。N-取代哌啶化合物CL-61917和CL-385319对H1和H2亚型禽流感有抑制活性,IC50达到μM水平。分析化合物的耐药病毒株发现HA蛋白序列有氨基酸突变,这个氨基酸位于HA三聚体茎部区域靠近HA2融合肽的部位。BMY-27709,能够抑制A/WSN/33病毒和H1亚型和H2亚型的复制,IC50在3-8μM。对BMY-27709耐药的毒株HA基因序列分析发现了耐药突变位点位于HA2亚基的N端,表明化合物是结合于HA2亚基N端来阻止血凝素介导的膜融合。Stachyflin能够有效地阻止H1亚型和H2亚型禽流感的感染,IC50达到μM水平。对Stachyflin耐药的毒株HA基因序列分析发现了两个点突变,K51R、K121E,位于HA2亚基。CR6261单抗是新近发现的广泛中和抗体,对多种亚型的流感病毒均有效,在动物实验中能保护小鼠抵抗致死量病毒的攻击。晶体结构显示,它们都是结合于HA2上的茎部区域,使HA在酸性条件下保持中性状态的结构,阻止了病毒的进入。
以上研究表明血凝素的HA2亚基是一个潜在的小分子药物靶点和疫苗靶点。但是上述的有抑制流感病毒活性的化合物只限于特定的亚型,目前还没有对高治病性禽流感H5N1有抑制活性的报道。并且上述化合物的耐药株的突变位点都位于HA2亚基的茎部区域。这个区域分布着大量的可电离氨基酸,在病毒的进入过程中起着重要的作用。突变研究发现其中一些氨基酸残基会影响HA三聚体的稳定性,使融合PH升高。目前只获得了TBHQ与HA的共结晶,研究清楚了其抗病毒的机制。但其他化合物与血凝素结合的模式,抑制病毒进入的机制还不清楚。
目的和意义：在早前的研究中,我们发现了CL-385319对H5N1A/Vietnam/1194/2004有抑制作用,并且初步确认了靶点是血凝素的HA2亚基。本研究中旨在建立多种具有代表性毒株的高致病性禽流感H5N1假病毒,用于进一步确认CL-385319的抗H5N1作用和筛选其他小分子化合物；利用点突变技术往HA上引入突变位点,观察突变位点和CL-385319抑制活性的关系,确认CL-385319在血凝素上的作用位点；深入研究CL-385319与血凝素结合位点的结合模式,探讨CL-385319阻止流感病毒进入的机制；并以CL-385319为先导化合物进行结构改造得到一系列衍生物,评价衍生物的抗H5N1禽流感活性,寻找活性更好的化合物,确认衍生物的作用位点,初步总结构效关系,为进一步的药物结构优化和改造提供了空间。
方法和结果：实验数据均采用Mean±SD表示,采用SPSS13.0软件进行数据分析。先进行方差齐性检验,方差齐时采用one-way ANOVA方法进行显著性检验统计分析,多重比较采用LSD方法分析；方差不齐时采用近似F检验的Welch方法进行显著性检验统计分析,多重比较采用Dunnetf’s T3进行分析。p<0.05为差异有统计学意义。
作为先导化合物必须要有广谱的抗病毒活性,首先我们要建立多种具有代表性毒株的高致病性禽流感H5N1假病毒。我们将表达HA和NA的质粒,与HIV包膜蛋白基因缺失的骨架质粒pNL4-3.luc.R-E'共同转染293T细胞,收获上清,得到具有单轮感染活性的假病毒颗粒。本文一共构建了5种人源H5N1假病毒,分别是A/Qinghai/59/2005, A/Xinjiang/1/2006、A/Anhui/1/2005、A/HongKong/156/1997及A/Thailand/Kan353/2004。这5株H5N1假病毒都能成功感染MDCK细胞。在不同的药物浓度下,我们检测了CL-385319对5株H5N1假病毒的抑制活性。结果表明,CL-385319对5种H5N1假病毒均有抑制作用,并特异性作用于病毒的进入环节。对A/Qinghai/59/2005、A/Xinjiang/1/2006, A/Anhui/1/2005、A/Hong Kong/156/1997及A/Thailand/Kan353/2004毒株的IC50分别是1.5±0.13μM、2.22±0.24μM、0.37±0.12μM和2.62±0.08μM.这表明CL-385319具有广谱的抗H5N1禽流感活性。我们对这5株毒株的HA蛋白序列进行了对比,发现HA1段差别较大,HA2段同源性高,这符合我们早前的实验结果,CL-385319的作用位点在HA2亚基上。
要阐明CL-385319的作用机制,必须要明确化合物的具体作用位点。前人发现CL-385319有抑制H1亚型流感的活性,并且发现了HA2的两个突变N502D, F1102S能让H1亚型流感毒株产生耐药性。根据HA的同源性分型,H1,H5亚型流感属于同一组别,H3亚型属于另一组别。通过序列比对,我们发现H1N1与H5N1的HA2序列同源性达到78.5%,并且H5N1的HA2同样具有N502和F1102两个残基。利用SYBYL7.3软件的子程序Surflex-dock在H5N1血凝素的F1102残基处发现了一个空腔,位于血凝素的茎部区域。这个空腔是由Gl2、L22、K51、E1052、R106、T1072、L1082、D1092、F1102、T221、M241和E251氨基酸残基构成。
为了明确CL-385319在HA上的具体作用位点,我们以A/Qinghai/59/2005-HA质粒为模板,引入单点突变位点：包括M241A、E251A、 R3221A、K432A、D462A、G472A、V482A、N502D、K512A、E1052A、R1062A、 T1072A、pD1092A和F1102S。将鉴定为含有目的突变基因的阳性克隆质粒和NA质粒,与HIV包膜蛋白基因缺失的骨架质粒pNL4-3.luc.R-E共同转染293T细胞包装出假病毒颗粒。我们测定了14个突变位点假病毒感染MDCK细胞的荧光发光值。Levene方差齐性分析检验结果是,F=4.039,P<0.000,方差不齐(P<0.05)；采用近似F检验的Welch方法,F=3460.069,P<0.000,采用Dunnett'sT3检验各种点突变与野生型(WT)的差异,发现G472、K512A、R1062A、D1092A与WT没有显著性差异,P值分别是(0.951,0.857,1.000,1.000),其余突变假病毒都与野生型有显著性差异,P值均<0.000。而且带有N502D、E1052A、T1072A点突变的假病毒与Blank没有显著性差异,P值均大于0.05,这表明假病毒完全丧失感染能力；而F1102S假病毒感染性较低,与野生型假病毒有显著性差异,P<0.000,这表明HA2的N502、E1052、T1072、F1102对病毒的进入起着关键的作用。接着在不同的浓度下,我们检测了突变前后的假病毒对CL-385319的敏感性。结果发现,V482A、M241A、F1102A这三个点突变假病毒均对CL-385319耐药。E251A、D1092A突变的假病毒敏感性增强。其他位点的突变假病毒对CL-385319敏感性变化均不明显。为了进一步确认假病毒感染力丧失／降低以及耐药性是由氨基酸突变引起的,我们用ELISA检测了突变假病毒裂解液中P24的含量,Levene方差齐性分析检验结果是F=1.545,P=0.155,方差齐性,采用one-way ANOVA方法,F=0.633,P=0.816,突变组与野生型组没有显著性差异,这表明突变假病毒与野生型的P24抗原含量在同一个水平,所有带有突变的假病毒均包装成功。我们还用Western blot检测突变H5N1假病毒的血凝素表达量,发现N502D、E1052A、T1072A、V482A、M241A、F1102A的突变假病毒的血凝素都正常表达。这些结果表明CL-385319在HA上的作用位点是在HA的茎部区域,V482A、M241A、F1102A是CL-385319直接结合的氨基酸残基。我们进一步地分析了这些关键氨基酸在人源H5N1禽流感中的保守性,对目前已知的405列HA序列进行比对发现V482、M241、F1102、E1052和T1072都是高度保守的。
为了阐明CL-385319与血凝素结合位点的结合模式,探讨CL-385319阻止流感病毒进入的机制,我们利用分子对接和分子动力学模拟的方法进行了研究。我们首先用Autodock对接软件得到了CL-385319与HA三聚体对接复合物的初始结构,接着运用Amber软件进行了长达47ns动力学模拟,用MM GBSA计算了体系的结合自由能,用丙氨酸扫描方法预测了与CL-385319作用的关键残基,用量化计算软件Laplacian计算了CL-385319与靶穴残基间氢键的相互作用。我们对动力学模拟中能量最低点的构象进行了分析。CL-385319结合位点在HA三聚体的茎部区域,由相邻的两个HA单体构成MM_GBSA结果显示复合物的结合自由能是-40.6109kcal mol-1,这表明结合过程是自发进行的.CL-385319通过π-π相互作用嵌合在HA2的F1102和HA1的M241氨基酸残基之间,形成一个三明治的结构,并且与M241、G12、R1062、T1072形成氢键。π-π相互作用和氢键作用对CL-385319与HA形成稳定的复合物起了主要作用。
我们比对了动力学模拟过程中HA的平均结构和HA的晶体结构(pdb:2IBX),发现HA的晶体结构中原本不存在结合的靶穴,在初始结构中,F1102和M241之间的距离不足以让CL-385319的苯环部分插进去。而在动力学模拟过程的平均结构中,我们发现靶穴的氨基酸残基位置的移动都在RMSD的范围内除了M241的位置发生了明显变化,M241残基朝着F1102相反的方向往外移动,使F1102和M241之间的距离增加到8.001A,产生了一个可供CL-385319的苯环部分结合的空腔,最终形成三明治结构。在分析MD过程中的结构时还发现V482残基对CL-385319与HA结合起着关键的作用。在MD的早期构象中,CL-385319的三氟甲基与V482形成了三个氢键,起到稳定的作用,而此时F1102和M241之间的距离还没有增加。随着MD过程的进行,三氟甲基与V482的氢键断裂,F1102和M241的距离增加,与CL-385319的苯环形成三明治结构,体系结合自由能降低。这些计算结果表明CL-385319在与HA的结合中,HA结合位点的氨基酸残基位置发生了变化来与CL-385319结合,这符合Koshland提出的诱导契合学说。
CL-385319的关键结合位点位于血凝素的茎部区域,血凝素只有这个区域的可电离氨基酸会因水解时融合肽的插入而被深埋入三聚体的内部,两者之间的氢键作用和盐桥作用在中性状态下能稳定HA1/HA2的结构。在酸性条件下,氢离子增加,这些可电离氨基酸的质子化状态改变,使HA进入融合态结构。前人的突变研究也显示这些残基与低pH触发的HA结构重组直接相关。E1052K突变能增加血凝素在低pH下的稳定性,而R1062H突变增强了H2亚型血凝素在低pH下的稳定性,并且阻止了HA发生融合结构重组。然而这个区域的氨基酸残基从来没有明确地与病毒进入抑制剂联系起来,因此弄清楚CL-385319稳定血凝素的机制会对新的进入抑制剂设计带来启发。E1032,E1052,R1062和T1072位于HA茎部区域。E1052A和T1072A单点突变使H5N1假病毒丧失了感染能力。分子动力学模拟显示CL-385319与R1062和T1072发生氢键相互作用,稳定了这个节段的构象,可能改变了这些残基的质子化状态,阻止了酸性破坏残基间的电荷作用。研究报道融合肽的G12E突变能够使膜融合过程不能进行,CL-385319能与G12形成氢键,对融合肽起到稳定的作用,阻止其移动。另外,HA1与HA2亚基的分离是HA结构重组的第一步。我们的动力学模拟结果表明CL-385319的苯环与HA1亚基上的M24和HA2亚基上的F110由于π-π作用形成三明治结构,起到固定HA1和HA2亚基的作用。综上所述,在融合pH时,CL-385319通过稳定HA的中性结构,阻止了病毒进入过程中所必需的结构重组,起到抗病毒的作用。
我们以CL-385319为先导化合物进行结构修饰,合成了一系列衍生物。我们利用假病毒体系评价了衍生物抗H5N1的活性,一共筛选出26个活性化合物,其中有4个活性化合物达到nM水平,活性最好的化合物是3-氟-5-三氟甲基-N-((2-噻吩基)乙基)苯甲酰胺。为了确定新化合物的作用位点,我们检测了这26个衍生物对V482A、F1102S和M241A这三种H5N1突变体假病毒的敏感性。结果显示这26个衍生物对这三株假病毒都没有抑制活性,这表明衍生物是作用于CL-385319一样的作用位点,也就是血凝素的茎部区域。其抗病毒的机理也是稳定HA的中性结构,阻止了酸性低pH触发的血凝素结构重组,从而阻止膜融合和病毒进入。我们测定了活性较好的衍生物A11、A12、A13、A14对神经氨酸酶的抑制活性,Levene方差齐性分析检验结果是F=2.123,P=0.115,采用One-way ANOVA方法,F=960.989,P=0.000,有显著性差异,用LSD进行多重比较,分析化合物组与No compound组的差异,发现A1l、A12、A13、CL-385319与No compound组没有显著性差异,P值均大于0.05,这表明A11、A12、A13、A14化合物对神经氨酸酶没有抑制活性。
我们初步总结了26个衍生物的构效关系。我们发现衍生物的立体构型与CL-385319越接近的,活性越好。我们发现化合物母核上的取代基与活性有明确的关系。针对R4基团,F取代CF3或CF3取代H都能增强抗流感活性。T基团的空间位阻越大活性越差.空间位阻可能影响了化合物与R1062,T1072和M241残基形成氢键作用。化合物的苯甲酰胺的氧原子与G1残基形成氢键,对活性很关键,把甲酰胺取代成-SO2NH2和-COO-,化合物的活性则明显下降。
结论
1.CL-385319具有广谱的抗H5N1禽流感活性,对A/Qinghai/59/2005、 A/Xinjiang/1/2006、A/Anhui/1/2005、A/HongKong/156/1997和A/Thailand/Kan353/2004都有很强的抑制作用。
2.CL-385319的作用位点位于H5N1的HA的茎部区域的保守空腔,由G12、L22、K512、E1052、R1062、T1072、L1082、D1092、F1102、T221、 M24和E251氨基酸残基构成,其中V482A、M241A、F1102A是CL-385319直接结合的氨基酸残基。
3.CL-385319与HA的结合是一个诱导契合的过程。
4.CL-385319与HA的结合,稳定了HA的中性结构,阻止了pH触发的融合结构重组,阻止了禽流感病毒的进入。
5.以CL-385319为先导化合物合成的一系列衍生物具有抗H5N1和H1N1禽流感病毒活性,验证了药物靶点的真实性,为进一步化合物结构优化和改造提供了空间。
Background:Influenza continues to pose serious threats to public health worldwide since seasonal influenza epidemics can affect up to15%of the population and result in more than500,000deaths worldwide each year, while some influenza pandemics, like the1918flu pandemic (the Spanish Flu), may lead to millions of deaths. In2009, a new influenza pandemic caused by a novel swine-origin influenza A virus (S-OIV) H1N1has resulted in millions of infections in more than213countries. Highly pathogenic avian influenza (HPAI) H5N1viruses have caused350fatal cases among a total of596infected individuals, with a case-fatality rate of59%. The HPAI H5N1virus emerged as a human pathogen in1997within the Hong Kong. Since late2003, the H5N1virus has expanded its geographical range to affect poultry in East and Southeast Asia. Subsequently, H5N1virus has spread to wild birds across Eurasia and as far west as England and Africa. Since viruses are passaged in a particular host they can adapt to that host by mutating the receptor-binding site in the viral HA, there is a growing concern about H5N1acquiring human-to-human transmissibility.
At the current time, only2classes of influenza antiviral agents are currently licensed M2ion channel inhibitors and neuraminidase inhibitors. The M2ion channel inhibitors (amantadine and rimantadine) are ineffective against influenza B strains and, influenza A virus resistant to amantadine and rimantadine can emerge quickly during treatment. Since2003, the M2ion channel inhibitors are recognized to have extremely limited clinical utility because ofworldwide influenza A virus resistance to these2drugs. Neuraminidase inhibitors are both effective against influenza A and B strains. But clinical virus resistant to oseltamivir has been reported.
Entry inhibitors, also known as fusion inhibitors, are a class of antiretroviral drugs, used in combination therapy for the treatment of HIV infection. This class of drugs interferes with the binding, fusion and entry of virus to host cells. HA plays crucial roles in the early stage of influenza virus infection, including virus binding to host receptors, viral entry, and membrane fusion. It is a potential target for entry inhibitors.
Several small molecules that can block the pH-depended irreversible conformational changes of HA, such as TBHQ, BMY-27709, CL-61917, CL-385319Stachyflinwere shown to inhibit IAV infection. TBHQ can inhibit H3-subtype influenza virus, but not H5-subtype. TBHQ could induce drug-resistance mutations in HA2subunit, suggesting that it acts on the HA protein. X-ray crystallographic study indicates that TBHQ binds with HA2and locks the HA2trimer at the neutral-pH conformation, through the interaction of TBNQ with three ionizable amino acids in this site:Arg-54, Glu-57and Glu-97from HA2subnuit. CL-61917and CL-385319show inhibitory activity against H1and H2subtypes of IAVs with IC50at low micromolar levels. Analysis of HA genes of the mutant viruses resistant to these compounds showed that amino acid mutations clustered in the stem region of the HA. trimer in and near the HA2FP. BMY-27709can inhibit H1and H2subtype IAV infection through its specific interaction with the HA protein to repress its conformational change of HA induced by low-pH. Sequence analyses of the HA gene of the variants resistant to BMY-27709mapped the amino acidsubstitutions responsible for drug resistance to a region located near the N terminus of HA2. Stachyfl in is effective in inhibiting infection by H1and H2subtype IAVs with IC50at low μM level. It was shown that Stachy fl in interfered with low pH-induced conformational change of HA, thereby blocking HA-mediated virus-cell fusion. Analysis of the HA gene sequences of the IAV variants with resistance to Stachy fl in revealed that two amino acid substitutions, K51R and K121E, in the HA2subunit of the HA protein. Recent studies have indicated that monoclonal antibody(mAb) CR6261, which recognizes a highly conserved helical region in the membrane-proximal stem of HA1and HA2, could neutralize the virus by blocking conformational rearrangements associated with viral membrane fusion. However, until now, these ionic residues had not been correlated with properties of influenza fusion inhibitors. Until now, only the TBHQ antiviral mechanism has been resolved by X-ray crystallography. The mechanism of action of other entry inhibitors has not been uncovered yet.
These studies demonstrated that HA2subnuit, especially the stem region, to be an antiviral target for entry inhibiots and for inducing broad neutralizing antibody responses. But the molecules mentioned above just showed subtype-dependent activities, and small-molecule inhibitors which inhibit HPAI H5N1have not been reported so far. In addition, Ionic residues at or near the stem region are suggested to play a critical role in fusion activation because of the apparent relationship between their chemical environment and HA sensitivity to low pH. Mutational analysis showed that amino acids in this cavity are associated with low pH-induced conformational change.
Objection:We previously found that CL-385319can inhibit A/Vietnam/1194/2004H5N1in vitro and might target on HA2subunit. The present study aims to establish different isolate H5N1pseudovirus systems to confirm CL-385319antiviral activity and to screen other small-molecule compounds. To identify the critical binding site of CL-385319on HA by detailed site-directed mutagenesis. To uncover the molecular mechanism underlying the action of CL-385319by extensive computational simulations, this could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza. A series of derivatives which focused on structural variation of CL-385319were synthesized and evaluated their anti H5N1activity. The structure-activity relationship is obtained for further research.
Methods and Results:The data are presented as means±standard deviations. Levene's test for homogeneity of variance is performed. When the assumption of equal variances holds, One-Way ANOVA is preferable to the F statistic and LSD is performed to do pairwise multiple comparisons. When the assumption of equal variances does not hold, Welch is preferable to the F statistic and Dunnett's T3is performed to do pairwise multiple comparisons.
To test whether CL-385319may inhibit different isolate of H5N1IAV, we generated five H5N1pseudoviruses. The plasmids encoding HA of different isolate H5N1IAV, including A/Thailand/Kan353/2004, A/Qinghai/59/2005, A/Xinjiang/1/2006, A/Anhui/1/2005, A/Hong Kong/156/1997, as well as the A/Thailand/Kan353/2004Nl subtype NA plasmid, were cotransfected with an HIV-1env deleted backbone plasmid pNL4-3.1uc.R-E-in293T cells. We harvested H5N1pseudovirus particles from the culture supernatants. These five H5N1pseudoviruses were able to infect MDCK and maintain significant high infectivity. CL-385319could inhibit these five H5N1pseudoviruses with IC50of4.00±0.38μM, 1.50±0.13μM,2.22±0.24μM,0.37±0.12μM,2.62±0.08μM, respectively. However, CL-385319had no effect on VSV-G pseudovirus infection, demonstrating that the compound specifically interferes with the entry of influenza virus. We compare the HA sequences of these five H5NA isolate and found that the amino acid sequence identity of HA1is releatively low, that of HA2is much higher. This is consistent with our previous study, the binding sites of CL-385319located in HA2.
It is needed to identify the critical residues for CL-385319binding first to uncover the detailed binding mechanism. Previous study showed that CL-385319is effective in inhibiting infection by HI subtype IAVs, but N502D, F1102S rendered the virus highly resistant to CL-385319. Based on phylogenetic analysis, H1and H5belong to Group1, while H3belongs to Group2of IAV HAs (Russell et al.,2008). The amino acid sequence identity of HA2subunit between H5and H1is81%. Specially, the critical residues for the activity of CL-385319, N502and F1102, identified through CL-385319-resistant H1N1virus, are identical between H5and H1or H2subtypes. Using Surlex-Dock program of SYBYL7.3, a cavity was found in the stem region of the HA near the fusion peptide. The cavity is surrounded by a few residues:G12, L22, K512, E1052, R1062, T1072, L1082, D1092, F1102, T22,, M241, E251and R3221.(1refers to HA1subunit,2refers to HA2subunit)
We identified the critical residues for CL-385319binding on H5N1hemagglutinin using site-directed mutagenesis analysis. We generated a series of mutant HA pseudoviruses based on A/Qinghai/59/2005-HA plasmid. The residues surrounded the cavity including:M24h E251, R3221, K432, D462, G472, V482, N502, N502, K512, E1052, R1062, T1072, D1092, and F1102were selected and substituted by alanine. The mutant pseudovirus susceptibility to CL-385319was tested. The result of Levene's test for homogeneity of variance is F=4.039, P<0.000. Welch is preferable to the F statistic, F=3460.069, P<0.000; Dunnett's T3is performed to do pairwise multiple comparisons. Results showed that the RLU of G472A, K512A, R1062A, D1092A mutations have no significance with that of WT, P=0.951,0.857,1.000,1.000, respectively. The rest of mutations have significance with WT, P<0.000. Results showed that the M241A, F1102S and V482A mutations rendered the virus highly resistant to CL-385319, suggesting that M241, F1102and V482are the critical residues involved in the binding of CL-385319. M241, F1102and V482are highly conserved in all H5N1strains. However, D1092A and E251A mutations resulted in increased sensitivity of pseudoviruses to CL-385319. One possible reason is that the mutations of E251A and D1092A cause changes of conformation and hydrophobicity of the cavity, which might increase the susceptibility of F1102and M241.to CL-385319. Interestingly, the individual N502D, E1052A and T1072A mutation eliminated pseudovirus infection, P>0.05vs Blank; F110S showed moderate infectivity, P=0.000vs WT, while Western blotting analysis showed that none mutation affected HA protein expression on the surface of pseudovirus. The amount of p24contained in the pseudovirus tested by HIV-1p24ELISA were on the same level. The result of Levene's test for homogeneity of variance is F=1.545, P=0.155. One-way ANOVA is preferable to the F statistic, F=0.633, P=0.816. There is no significance between mutation group and Wildtype. These results suggest that N502, E1052, T1072and F1102are critical for viral entry and that these mutations might interfere with the structural rearrangement needed for membrane fusion.
Furthermore, extensive computational simulations, including molecular docking, molecular dynamics (MD) simulations, MM_GBSA calculations, as well as charge density and its Laplacian calculations, have been carried out to uncover the detailed mechanism of CL-385319binding to HA. The CL-385319binding site is formed by HAl and HA2in one monomer. There are three identical binding sites per HAtrimer. The calculated binding free energy of the complex was-40.6109kcal mol-1, which indicated that the binding of CL-385319to HA could increase the stability of HA protein. Consistent with the experimental mutagenesis result, the π-π interactions between CL-385319and residues F1102and M241make important contributions to the binding affinity. In addition, CL-385319can form hydrogen bonds with the residues of E1052, R1062and T1072.
As suggested by Koshland, the induced-fit model describes the capability of a proper ligand to induce specific structural change of the active site such that the protein is able to achieve the final key-lock state.We found that the binding of CL-385319to HA is a process of "induced fit". In the CL-385319-HA complex structure derived from molecular docking, which was used as starting structure for molecular dynamics simulation, the trifluorophenyl group of CL-385319formed three important H-bonds interaction with V482-The averaged structure of47ns MD simulation shows that the benzene ring of CL-385319was sandwiched by F1102and M24] via π-π interactions, while the H-bonds interaction between trifluorophenyl group and V482was not observed in the lowest energy structure. Therefore, we believe that the H-bond interaction between the trifluorophenyl group and V482stabilizes the inhibitor in the early stage of MD and facilitates the formation of π-π interactions. Originally, no binding pocket was observed between F1102and V482. However, during MD simulation, a significant conformational change of the M24] residue was observed, as it shifted away from F1102to accommodate CL-385319. In addition, the free energy of CL-385319binding to HA calculated by the MM_GBSA method showed that the binding process is thermodynamically favorable. Therefore, we conclude that CL-385319binding to HA occurs through the induced-fit pathway.
The critical residues for CL-385319binding locate in the stem region of HA. Ionic residues at or near this region are the only residues that experience different solvent environments before and after the insertion of fusion peptide into the cavity at the trimer interface, a necessary priming step of HA fusion. These residues are therefore suggested to play a critical role in fusion activation because of the apparent relationship between their chemical environment and HA sensitivity to low pH. Mutational analysis showed that amino acids in this cavity are associated with low pH-induced conformational change. For example, the E1052K mutation increased the stability of HA protein in low-pH, while the R1062H mutation improved the stability of H2-typed HA and inhibited the irreversible conformational rearrangement at fusogenic pH14. However, until now, these ionic residues had not been correlated with properties of influenza fusion inhibitors. Therefore, understanding how CL-385319stabilizes HA could provide a clue for the development of new influenza fusion inhibitors. In H5N1-typed HA2, E1032, E1052, R1062and T1072are located in the stem region which experienced different environments as described above. Mutagenesis results showed that E1052A and T1072A mutation eliminated pseudovirus infectivity. Molecular dynamics simulation showed that CL-385319has hydrogen-bonding interactions with residues of R1062and T1072. These interactions might interfere with the disruptions of inter-subunit ionic interactions induced by low-pH. Meanwhile, CL-385319has hydrogen-bonding interaction with residues of G12, the first residue of HA fusion peptide. It is known that G12E mutation abolish HA-mediated mutation. CL-385319might stabilize the conformation of the fusion peptide through this interaction. In addition, the dissociation of HA1from HA2is the first step for HA conformational rearrangement. Previous study showed that CL-385319can inhibit the proteolysis of purified HI-typed HA in low-pH. We demonstrated that the π-π interaction between CL-385319and the residue of F1102and M24] may provide a cross-linking of the HA trimer, thus inhibiting the dissociation of HA1and HA2; even the HAO was proteolyzed into HA1and HA2. As a consequence, CL-385319binding stabilizes the neutral pH conformation of HA, rendering its membrane fusion inactive.
A series of derivatives which focused on structural variation of CL-385319were synthesized and evaluated for antiviral activities against H5N1influenza virus.26analogues showed antiviral activities, four of them were found to be highly potent with IC50at nM level. The most effective compound was3-fluoro-5-(trifuoromethyl)-N-(2-(thiophen-2-yl)ethyl)benzamide. All of the V482A, F1102A and M241A mutant H5N1were resistant to these analogues, which suggested that they bind the same side and adopt the same mechanism to against H5N1as CL-385319did. All, A12, A13, A14were tested for the activity of N1-typed neuraminidase. The result of Levene's test for homogeneity of variance is F=2.123, P=0.115. One-way ANOVA is preferable to the F statistic, F=960.989, P<0.000; LSD is performed to do pairwise multiple comparisons. There is no significance between compound group and No compound group, P>0.05. The result showed that A11,A12, A13, A14have no activity on N1-typed neuraminidase
We further analysed the structure-activity relationship. Those analogues which have similar steric structure with CL-385319show higher antiviral activities. We found that replacement of R4-CF3-with F-group or H-with CF3-enhances the inhibitory activity. The steric hindrance of T-receptor affect activities significantly, analogues with higher antiviral activities have less steric hindrance. This might be associated with the formation of hydrogen bonds between compounds and R1062, T1072and M241residues.
Conclusion
1. CL-385319has potent inhibitory activity against a broad range of H5N1sublineages, including A/Thailand/Kan353/2004, A/Qinghai/59/2005, A/Xinjiang/1/2006, A/Anhui/1/2005and A/Hong Kong/156/1997.
2. The binding cavity of CL-385319locates in the stem region of HA and, is surrounded by a few residues:G12, L22, K512, E1052, R1062, T1072, L1082, D1092, F1102, T22,, M241, E251and R3221. The critical residues for CL-385319binding are F1102, M24, and V482.
3. CL-385319binding to HA occurs through the induced-fit pathway.
4. CL-385319binding stabilizes the neutral pH conformation of HA, rendering its membrane fusion inactive.
5. CL-385319derivates have potential inhibitory activity against H5N1and H1N1influenza virus. This confirmed the existance of binding pocket and provided a valuable foundation for the structure-based design of more potent influenza fusion inhibitors
引文
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