| Background:Influenza A viruses (IAVs) are one of the major causative pathogens of human acute respiratory disease responsible for seasonal epidemics and reoccurring pandemics of influenza, which poses a significant threat to human health and economic development. So far, there are only two classes of drugs available for the treatment of influenza A virus infection:the matrix protein 2 (M2) inhibitors such as amantadine and rimantadine, and the neuraminidase (NA) inhibitors like oseltamivir and zanamivir. These clinically used drugs are functioned by blocking the proton channel activity of the influenza A viral M2 protein, or binding to NA to inhibit virus budding. However, due to the emergence of drug-resistant viral strains, new antiviral strategies, targeting other viral proteins or cellular factors involved in the influenza virus life cycle, are urgently needed.Influenza A viruses (IAV) are enveloped viruses antigenically consisting of two major surface proteins:hemagglutinin (HA) and neuraminidase (NA). The HA of IAV is a glycoprotein mediating the binding of influenza viron to host cells and entry of the viral genome into target cell. It is composed of HAl and HA2 two subunits with each ca.327 and 222 residues, respectively. The HAl and HA2 subunits are linked by a single disulfide bond to form a "mushroom" like structure. The globular head of the "mushroom" is constructed by HAl subunits responsible for the receptor binding, while the stem of the "mushroom" surrounded by the N-and C-terminal segments of HAl is composed of the trimeric HA2 subunits, and is responsible for the viral-endosomal membrane fusion. In the events of influenza A virus infection, HA1 binds to the a-2,3 or a-2,6 linked sialic acids in membrane glycoprotein receptor of host cell, following endocytosis for virus entry. Once within the endosomal compartment, the viron is exposed to the increasing acidic condition (pH 5-6) which triggers the conformational changes of HA protein, thereby promoting fusion of the viral and endosomal membranes. Upon fusion, the viral genetic materials are released into the host cell cytoplasm and the viral ribonucleoproteins (vRNPs) traffic to the nucleus, where viral replication is initiated. Thus, it can be concluded that the HA protein including HA1 and HA2 subunits is involved in the key steps of influenza virus life cycle and plays a critical role in the events of virus binding to host cells, entry and membrane fusion, consequently, is recognized as a promising drug target for anti-IAV agents to intervene.Chapter 1 A "building block" approach to the new influenza A virus entry inhibitors with reduced cellular toxicitiesObjectives:To develop novel antiviral agents as "entry inhibitors", this chapter proposes a promising approach to design new antiviral agents with high selectivity indices. We show a new class of anti-influenza virus peptides generated by conjugating two known short peptides:part-1 (named Jp with the sequence of ARLPR) and part-2 (named Hp with the sequence of KKWK). The new peptides are thus created by hybridization of these two domains at C-and N-termini, respectively. Herein, we report on the design, antiviral activity and mode of action of these peptides.Methods:a. All peptides were synthesized by using standard 9-fluorenylmethoxy carbonyl (Fmoc) solid phase protocol on Rink Amide MHBA resin. These lipopeptides were found to have a purity of or above 90%.b. The antiviral activities of peptides against influenza viruses were tested with CPE assay. The viral strains were including:influenza A/Aichi/2/68 (H3N2), A/Puerto Rico/8/34 (H1N1), A/FM/1/47 (H1N1) mouse adapted strain, A/Puerto Rico/8/34 (H1N1) with NA-H274Y mutation virus, clinical isolates of 690 (H3 subtype) and 699 (H3 subtype). In addition, to show the wide-spectrum of antiviral activities of these peptides, the influenza B virus was also tested.c. The antiviral activity of C20-Jp-Hp was further assessed by a series of assays, including Quantitative real-time PCR and Plaque reduction assays.d. The most active peptide of C20-Jp-Hp was subject to the extensive mechanism studies by adopting various approaches, such as Neuraminidase (NA) inhibition assay, Measurement of the inhibitory activity on the entry of H5N1 pseudovirus and VSVG pseudovirus, and various drug administrations, such as Preventive treatment, Pre-incubation treatment, Simultaneous treatment, and Post-treatment of virus assays.e. The antiviral mechanism study of C20-Jp-Hp:The HA inhibition (HI) assay was carried out to determine whether sialic acid binding site on HA1 subunit was the possible target of C20-Jp-Hp. To test that whether HA2 subunit was the possible target of C20-Jp-Hp, a hemolysis inhibition assay was employed to evaluate the inhibitory effect of C20-Jp-Hp on the lysis of erythrocyte induced by influenza virus of A/PR/8/34 (H1N1) under low pH. f. To confirm the antiviral mechanism study of C20-Jp-Hp, the circular dichroism (CD) spectra analyses and a computer-assisted modeling were performed.Results:a. The anti-IAV screening results identified that C20-Jp-Hp was the most potent peptide with IC50 value of 0.53 μM against A/Puerto Rico/8/34 (H1N1) strain. All of these new peptides display lower toxicities toward mammalian cells and higher therapeutic indices than their prototypes.On the basis of these results, the mechanisms of action of C20-Jp-Hp were then extensively investigated.b. The Pre-incubation and Simultaneous treatments were proved to be the most effective drug administrations studied with the plaque reduction assay. Thus, it can be concluded that C20-Jp-Hp exerts its antiviral effects in the early stage of viral infection. In addition, C20-Jp-Hp exhibited an apparent inhibitory effect on H5N1 pseudovirus, while no activity against VSVG pseudovirus. The NA inhibition assay was employed to evaluate the enzymatic activity of NA, which showed that no inhibitory effect in the test range from 0.39 to 50 μg/mL at all, suggesting that C20-Jp-Hp was unable to inhibit the NA activity. Overall, these data indicate that C20-Jp-Hp may interact with the enveloped glycoprotein of HA, thereby inhibiting the infectivity of influenza A viruses.c. The HA inhibition (HI) assay was carried out to determine whether sialic acid binding site on HA1 subunit was the possible target of C20-Jp-Hp. As a result, no inhibition of agglutination of 1%chicken erythrocytes was observed, indicating that sialic acid binding site on HA1 subunit was not the binding site of C20-Jp-Hp. Furthermore, the hemolysis inhibition assay showed that the lysis of erythrocytes under acidic condition was decreased in the presence of C20-Jp-Hp compared with the hemolytic effect of virus alone, suggesting that the antiviral effects of C20-Jp-Hp may be associated with the inhibition of the conformational rearrangements of HA2 subunit thereby interrupting the fusion of virus-host cell membranes.d. The CD spectroscopy indicated that the significant changes were happened upon addition of C20-Jp-Hp into HA-FP-O, especially at pH 5, indicating that C20-Jp-Hp may interact with the fusogenic region of HA2, thus supporting the notion that the HA2 subunit of the viral glycoprotein is the specific target of C20-Jp-Hp. This data was further confirmed by computer-assisted modeling, which indicated that C20-Jp-Hp may interact with the fusogenic region of HA2, by which to block the conformational change of HA2, and subsequently leading to the inhibition of virus entry.Conclusions:a. This study proposes a new approach to build the influenza A virus entry inhibitors. In addition to the potent and wide-spectrum antiviral activities, these newly created peptides also exhibit a much lower cellular toxicities and higher selectivity indices towards mammalian cells, thus displaying a promising potential in the development of new anti-IAV drugs, which will lead us to a more vigorous and extensive in vitro and in vivo study in the near future.b. C20-Jp-Hp was represented as the most potent peptide when tested with a wide variety of influenza viruses in comparison with other analogues. Thus, we chose C20-Jp-Hp as a lead compound for the following mechanism study in this thesis. The mechanism study indicated that C20-Jp-Hp may inhibit the viral infection in the early stage by interacting with the fusogenic region of HA2 subunit. This process involves the block of conformational rearrangements of HA2, thereby interfering with the membrane fusion of virus with targeting host cells.c. The fusion peptides of influenza A viruses are known for their conservative sequences and a crucial role in the fusogenic process, therefore, are a potential target for antiviral drugs to intervene.Chapter 2 Cholesterol as a "Trojan Horse" for the Potent Influenza A Virus Entry Inhibitors Targeting a Conserved Region of HemagglutininObjectives:In the second section of this thesis, we describe a new class of lipopeptides created by employing cholesterol as a "trojan horse" to conjugate with a short anti-IAV peptide of KKWK. Besides the new class of influenza A virus entry blockers, this study also provides a novel strategy in designing potent anti-IAV agents.Methods:a. All peptides were synthesized by using standard 9-fluorenylmethoxy carbonyl (Fmoc) solid phase protocol assembled on Rink Amide MHB A resin. All synthesized peptides were showed to have a purity of or above 90%.b. The antiviral activities of peptides against influenza viruses were tested with CPE assay.c. The antiviral activitie of S-KKWK were further confirmed by a series of assays, including Quantitative real-time PCR assay and Fluorescence confocal microscopy assay.d. The most active peptide of S-KKWK were subject to the extensive mechanism studies by using various approaches, such as Neuraminidase (NA) inhibition assay, Measurement of the inhibitory activity on the entry of H5N1 pseudovirus and VSVG pseudovirus. In addition, various drug administration approaches were also employed.e. The antiviral mechanism of S-KKWK:We employed an HA inhibition (HI) assay to determine whether sialic acid binding site on HA1 was the possible target of S-KKWK. Second, a hemolysis inhibition assay was used to determine whether HA2 subunit was the possible target of S-KKWK.f. The combination of surface plasmon resonance (SPR) spectroscopy, circular dichroism (CD) spectroscopy and computer-aided docking simulation was next used to predict the possible binding site for S-KKWK.g. To determine if S-KKWK stabilizes HA native structure in both neutral and acidic conditions, sensitivity of HA to trypsin digestion was tested in which both HA protein resulting from neutral or a low pH treatment was cleaved by trypsin. The enzymatic digestion products were monitored on SDS-PAGE gel stained with Coomassie blue. Next, we inspected that whether the anti-IAV effect was due to the direct virolysis or virucidal activity of S-KKWK. After pre-treatment of influenza A/Puerto Rico/8/34 (H1N1) virus with S-KKWK for 30 minutes, the viral titer reduction was measured.Results:a. The anti-IAV screening results identified that S-KKWK was the most potent peptide with IC50 value of 0.7 μM against A/Puerto Rico/8/34 (H1N1) strain. Besides the potent activities against influenza viral strains such as H1N1 and H3N2 viruses, S-KKWK was also active against neuraminidase inhibitor-resistant strain and clinically relevant isolates with IC50 values ranging from 0.7 to 3.0 μM.b. Given the fact that the Pre-incubation and Simultaneous treatments were the most effective drug administrations tested with the CPE assay and confocol microscopy, it can be inferred that S-KKWK exerts antiviral effects in the early stage of viral infection. This result was further assisted by the results that S-KKWK exhibited an apparent inhibitory effect on H5N1 pseudovims, while no activity against VSVG pseudovims. Furthermore, the neuraminidase (NA) inhibition assay showed that no enzymatic inhibitory effect of S-KKWK towards NA was observed in the test concentration at all. Thus, combining together, these data indicate that S-KKWK interact with the enveloped glycoprotein of HA, by which to inhibit the infectivity of influenza A viruses.c. The SPR results indicated that the affinity constant (KD) of S-KKWK calculated from interactions between HA and S-KKWK was as 5.35 nM, confirming the presence of strong interactions between HA and S-KKWK. The HI assay results indicated that no apparent inhibition of agglutination of chicken erythrocytes was observed in the test range, thus excluding the sialic acid binding site on HA1 as the possible target of S-KKWK. In the presence of S-KKWK, the hemolytic effect under acidic condition was apparently decreased in comparison with the virus control, suggesting that the anti-IAV activity of S-KKWK may be associated with the lock of conformational changes of HA2 subunit, by which to interrupt the fusogenic process of virus-host cell membranes. Furthermore, the affinity constant (KD) obtained from SPR spectroscopy was as 16.3 nM, confirming the presence of strong interactions between HA2 subunit and S-KKWK.d. The CD curves did not exhibit any significant changes between the interactions of S-KKWK and HA-FP-O, a segment of peptide derived from the N-terminal fusion domain of HA2 subunit, indicating that the N-terminal region of fusion peptide of HA2 was excluded as the possible binding site of S-KKWK. Computer-aided simulation was next performed to predict the possible binding site for S-KKWK. As a consequence, a very conserved hydrophobic "pocket" formed by residues of Ile48, Val55, Ile56, Leu99, Val100, and Leu108 on HA2 subunt was observed, by which strong hydrophobic interactions between these residues and cholesterol moiety of S-KKWK would occur.Conclusions:a. It is noteworthy that in addition to the broad activities against those circulating influenza viral strains such as H1N1 and H3N2 viruses, S-KKWK and its derivatives are also effective against neuraminidase inhibitor-resistant influenza A virus, indicating a different mechanism from neuraminidase inhibitors in their killing effects. As a deduction, S-KKWK may inhibit the viral infection in the early stage by interacting with the HA, thus belonging to the virus entry inhibtor.b. Rather than interacting with HA1 subunit by inhibiting the adsorption of virus to the host cells, S-KKWK may bind to HA2 subunit to interrupt the conformational changes of HA2, by which the entry of influenza A viruses was subsequently blocked. These effects can be attributed to a conserved "hydrophobic pocket" on HA2 as the possible binding site of S-KKWK.c. The amino acid residues where the "hydrophobic pocket" formed were also shown in other influenza A virus subtypes and were very conserved. Therefore, it can be used as a potential drug target for designing novel anti-IAV molecules with broad protection against influenza A viral infections. |
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