New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC₅₀] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC₅₀] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM² % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.     

A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1

Membrane fusion is central to the entry of influenza virus into host cells. To quantitatively determine the fusion activity of hemagglutinin (HA) of avian influenza virus H5N1, we established a cell fusion assay based on a dual luciferase reporter gene. The HA fusion activity was assayed by measuring luciferase expression in fused cells, allowing a rapid, sensitive, and quantitative comparison of HA fusion activities at various pHs and in different cells types. The simplicity and the quantitative nature of this novel assay are ideally suited for identifying viral receptors or screening for inhibitors of viral entry in the future.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Human monoclonal antibodies to pandemic 1957 H2N2 and pandemic 1968 H3N2 influenza viruses

Investigation of the human antibody response to the 1957 pandemic H2N2 influenza A virus has been largely limited to serologic studies. We generated five influenza virus hemagglutinin (HA)-reactive human monoclonal antibodies (MAbs) by hybridoma technology from the peripheral blood of healthy donors who were born between 1950 and 1968. Two MAbs reacted with the pandemic H2N2 virus, two recognized the pandemic H3N2 virus, and remarkably, one reacted with both the pandemic H2N2 and H3N2 viruses. Each of these five naturally occurring MAbs displayed hemagglutination inhibition activity, suggesting specificity for the globular head domain of influenza virus HA. When incubated with virus, MAbs 8F8, 8M2, and 2G1 each elicited H2N2 escape mutations immediately adjacent to the receptor-binding domain on the HA globular head in embryonated chicken eggs. All H2N2-specific MAbs were able to inhibit a 2006 swine H2N3 influenza virus. MAbs 8M2 and 2G1 shared the V(H)1-69 germ line gene, but these antibodies were otherwise not genetically related. Each antibody was able to protect mice in a lethal H2N2 virus challenge. Thus, even 43 years after circulation of H2N2 viruses, these subjects possessed peripheral blood B cells encoding potent inhibiting antibodies specific for a conserved region on the globular head of the pandemic H2 HA.     

共表达甲型流感病毒M1和HA双基因的重组腺病毒的构建及鉴定

以我国分离的首株人H5N1亚型禽流感病毒(A/Anhui/1/2005)作为研究对象,通过引入内部核糖体进入位点序列(IRES),构建共表达H5N1亚型禽流感病毒膜蛋白基因M1和HA的重组腺病毒。PCR扩增禽流感病毒H5N1亚型M1和HA基因的全长可读框片段,先后亚克隆入pStar载体,然后扩增M1-IRES-HA片段并将其插入穿梭载体pShuttle-CMV,再与pAd-Easy载体在BJ5183菌中通过同源重组产生重组腺病毒载体,转化293细胞,包装出重组腺病毒Ad-M1/HA。将Ad-M1/HA感染293细胞,可观察到明显细胞病变效应,用免疫荧光及Western-blot方法均检测到M1和HA基因的表达。共表达M1和HA双基因的重组腺病毒的成功构建为开发新型重组腺病毒流感疫苗奠定了基础。     

Inhibition of Influenza H7 Hemagglutinin-Mediated Entry

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50∼6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound.     

Influenza A virus neuraminidase limits viral superinfection

Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of alpha-2,3- and alpha-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.     

Clioquinol targets zinc to lysosomes in human cancer cells

A panel of 52 murine monoclonal antibodies was found to recognize antigenic determinants that had been conserved among all major genetic subgroups of the H5N1 avian influenza virus prevalent since 1997. We screened a phage display library for peptides recognized by one such antibody (8H5). We analysed the specificity of 8H5 for reactive peptides presented as fusion proteins of HBc (hepatitis B core protein) and HEV (hepatitis E virus) structural protein, p239. This was then related to the specificity of the native HA (haemagglutinin) molecule by virtue of the capacity of fusion proteins to compete for 8H5 binding with different strains of H5N1 virus and the reactivity of antisera generated against fusion proteins to bind native HA molecules, and to inhibit haemagglutination and arrest infection by the virus. Nine reactive peptides of different amino acid sequences were identified, six of which were also reactive with the antibody in association with HBc and four were in association with p239. Binding occurred with the dimeric form of the four p239-fusion proteins and one of the HBc-fusion proteins, but not with the monomeric form. The HBc-fusion proteins blocked 8H5 binding with four strains of H5N1 influenza virus. Mouse antisera generated against fusion proteins bound to HA molecules, but did not inhibit haemagglutination or arrest H5N1 infection. Our findings indicate that 8H5 recognizes discontinuous sites presented by secondary and possibly higher structural orders of the peptides in spatially favourable positions for binding with the antibody, and that the peptides partially mimic the native 8H5 epitopes on the H5N1 virus.     

New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides

Influenza A viral (IAV) fusion peptides are known for their important role in viral-cell fusion process and membrane destabilization potential which are compatible with those of antimicrobial peptides. Thus, by replacing the negatively or neutrally charged residues of FPs with positively charged lysines, we synthesized several potent antimicrobial peptides derived from the fusogenic peptides (FPs) of hemagglutinin glycoproteins (HAs) of IAV. The biological screening identified that in addition to the potent antibacterial activities, these positively charged fusion peptides (pFPs) effectively inhibited the replication of influenza A viruses including oseltamivir-resistant strain. By employing pseudovirus-based entry inhibition assays including H5N1 influenza A virus (IAV), and VSV-G, the mechanism study indicated that the antiviral activity may be associated with the interactions between the HA2 subunit and pFP, of which, the nascent pFP exerted a strong effect to interrupt the conformational changes of HA2, thereby blocking the entry of viruses into host cells. In addition to providing new peptide “entry blockers”, these data also demonstrate a useful strategy in designing potent antibacterial agents, as well as effective viral entry inhibitors. It would be meaningful in treatment of bacterial co-infection during influenza pandemic periods, as well as in our current war against those emerging pathogenic microorganisms such as IAV and HIV.     

共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝     

Inhibition of influenza A virus (H1N1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin

Hemagglutinin (HA) of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin.     

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.     

Entry Properties and Entry Inhibitors of a Human H7N9 Influenza Virus

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC₅₀ of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.     

我国首例孕妇感染高致病性禽流感(H5N1)的病原学研究

对2005年11月8日安徽省铜陵市人民医院报告的一例孕妇不明原因肺炎病例的死亡病因进行研究。采集病人的气管吸出物及血液标本,用RT-PCR和Real-ti me PCR方法检测流感病毒A/M、A/H5N1、A/H7N7、A/H9N1亚型特异性核苷酸片段;气管吸出物接种SPF鸡胚进行病毒分离,并对分离物进行鉴定和序列测定及分析;利用血凝抑制试验检测血清标本抗体。结果表明病人气管吸出物可以检测到甲型流感病毒M片段及H5亚型的特异性HA基因。2005年11月9日采集的血清标本用Real-ti me PCR检测到甲型流感病毒M基因。从病人的气管吸出物中分离到H5N1病毒(A/Anhui/1/2005),对病毒的HA基因序列结果进行分析表明病毒是禽源的,其主要依据是受体结合位点第226~228位氨基酸位点(QSG)为禽流感病毒所特异,HA受体连接肽仍为9个碱性氨基酸(LRERRRKRP);基因进化树分析显示,HA基因与禽源病毒进化距离接近。发病后7、8、9d的血清H5N1禽流感病毒HI抗体小于20。对该病例的病原学研究证明,该病例为H5N1禽流感感染病例。     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Highly conserved M2e and hemagglutinin epitope-based recombinant proteins induce protection against influenza virus infection

Highly pathogenic influenza viruses continue to cause serious threat to public health due to their pandemic potential, calling for an urgent need to develop effective, safe, convenient, and universal vaccines against influenza virus infection. In this study, we constructed two recombinant protein vaccines, 2H5M2e-2H7M2e-H5FP-H7FP (hereinafter M2e-FP-1) and 2H5M2e-H5FP-2H7M2e-H7FP (hereinafter M2e-FP-2), by respectively linking highly conserved sequences of two molecules of ectodomain of M2 (M2e) and one molecule of fusion peptide (FP) epitope of hemagglutinin (HA) of H5N1 and H7N9 influenza viruses in different orders. The Escherichia coli-expressed M2e-FP-1 and M2e-FP-2 proteins induced similarly high-titer M2e-FP-specific antibodies in the immunized mice. Importantly, both proteins were able to prevent lethal challenge of heterologous H1N1 influenza virus, with significantly reduced viral titers and alleviated pathological changes in the lungs, as well as increased body weight and complete survivals, in the challenge mice. Taken together, our study demonstrates that highly conserved M2e and FP epitope of HA of H5N1 and H7N9 influenza viruses can be used as important targets for development of safe and economical universal influenza vaccines, and that the position of H7N9 M2e and H5N1 HA epitope sequences in the vaccine components has no significant effects on the immunogenicity and efficacy of M2e-FP-based subunit vaccines.     

甲型H5N1流感病毒M2与HA双基因载体疫苗在小鼠体内的免疫学评价

高致病性H5N1亚型禽流感病毒 (AIV) 严重威胁到人类健康,因此研制高效、安全的禽流感疫苗具有重要意义。以我国分离的首株人H5N1亚型禽流感病毒 (A/Anhui/1/2005) 作为研究对象,PCR扩增基质蛋白2 (M2) 和血凝素 (HA) 基因全长开放阅读框片段,构建共表达H5N1亚型AIV膜蛋白基因 M2和HA的重组质粒pStar-M2/HA。此外,还通过同源重组以293细胞包装出表达M2基因的重组腺病毒Ad-M2以及表达HA基因的重组腺病毒Ad-HA。用间接免疫荧光 (IFA) 方法检测到了各载体上插入基因的表达。按初免-加强程序分别用重组质粒pStar-M2/HA和重组腺病毒Ad-HA+Ad-M2免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集血清用于检测体液免疫应答,末次免疫后14 d采集脾淋巴细胞用于检测细胞免疫应答。血凝抑制 (HI) 实验检测到免疫后小鼠血清中的HI活性。ELISA实验检测到免疫后小鼠血清中抗H5N1亚型流感病毒表面蛋白的IgG抗体。ELISPOT实验检测到免疫后小鼠针对M2蛋白和HA蛋白的特异性细胞免疫应答。流感病毒M2与HA双基因共免疫的研究,为研究开发新型重组流感疫苗奠定了基础。     

Design,synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC₅₀ values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.     

Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones

The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC₅₀ value against influenza A/H3N2 virus of 1 nM and selectivity index of almost 2000. Resistance data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to achieve superior inhibitors of HA-mediated fusion.