Identification of potential inhibitors of coronavirus hemagglutinin-esterase using molecular docking, molecular dynamics simulation and binding free energy calculation.

The pandemic outbreak of the Corona viral infection has become a critical global health issue. Biophysical and structural evidence shows that spike protein possesses a high binding affinity towards host angiotensin-converting enzyme 2 and viral hemagglutinin-acetylesterase (HE) glycoprotein receptor. We selected HE as a target in this study to identify potential inhibitors using a combination of various computational approaches such as molecular docking, ADMET analysis, dynamics simulations and binding free energy calculations. Virtual screening of NPACT compounds identified 3,4,5-Trihydroxy-1,8-bis[(2R,3R)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl]benzo[7]annulen-6-one, Silymarin, Withanolide D, Spirosolane and Oridonin as potential HE inhibitors with better binding energy. Furthermore, molecular dynamics simulations for 100 ns time scale revealed that most of the key HE contacts were retained throughout the simulations trajectories. Binding free energy calculations using MM/PBSA approach ranked the top-five potential NPACT compounds which can act as effective HE inhibitors.

Discovery of a potent dual inhibitor of wild-type and mutant respiratory syncytial virus fusion proteins through the modulation of atropisomer interconversion properties.

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.

Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Characterization of the inhibitory effect of an extract of Prunella vulgaris on Ebola virus glycoprotein (GP)-mediated virus entry and infection.

Currently, no approved antiviral therapeutic is available for treatment or prevention of Ebola virus (EBOV) infection. In this study, we characterized an EBOV-glycoprotein (GP) pseudotyped HIV-1-based vector system in different cell cultures, including human umbilical vein endothelial cells (HUVECs) and human macrophages, for the screening of anti-EBOV-GP agent(s). Based on this system, we demonstrated that an aqueous extract (CHPV) from the Chinese herb Prunella vulgaris displayed a potent inhibitory effect on EBOV-GP pseudotyped virus (EBOV-GP-V)-mediated infection in various cell lines, including HUVEC and macrophage. In addition, our results indicated that CHPV was able to block an eGFP-expressing Zaire ebola virus (eGFP-ZEBOV) infection in VeroE6 cells. The anti-EBOV activity of CHPV was exhibited in a dose-dependent manner. At a 12.5 µg/ml concentration, the CHPV showed a greater than 80% inhibition of EBOV-GP-V and eGFP-EBOV infections. Likewise, our studies suggested that the inhibitory effect of CHPV occurred by binding directly to EBOV-GP-Vs and blocking the early viral events. Interestingly, our results have shown that CHPV was able to enhance the anti-EBOV activity of the monoclonal antibody MAb 2G4 against EBOV-GP. Overall, this study provides evidence that CHPV has anti-EBOV activity and may be developed as a novel antiviral approach against EBOV infection.

Efficient reovirus- and measles virus-mediated pore expansion during syncytium formation is dependent on annexin A1 and intracellular calcium.

UNLABELLED: Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytium formation and are virulence determinants of the fusogenic reoviruses. While numerous studies on the FAST proteins and enveloped-virus fusogens have delineated steps involved in membrane fusion and pore formation, little is known about the mechanics of pore expansion needed for syncytiogenesis. We now report that RNA interference (RNAi) knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein-mediated pore expansion during syncytiogenesis but had no effect on pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Coimmunoprecipitation revealed calcium-dependent interaction between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen-specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of postfusion pore expansion and syncytium formation following virus-mediated cell-cell fusion. IMPORTANCE: Numerous enveloped viruses and nonenveloped fusogenic orthoreoviruses encode membrane fusion proteins that induce syncytium formation, which has been linked to viral pathogenicity. Considerable insights into the mechanisms of membrane fusion have been obtained, but processes that drive postfusion expansion of fusion pores to generate syncytia are poorly understood. This study identifies intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion but not membrane fusion and pore formation. This is the first demonstration that intracellular AX1 is involved in pore expansion, which suggests that the AX1 pathway may be a common host cell response needed to resolve virus-induced cell-cell fusion pores.

Water extract of Cinnamomum cassia Blume inhibited human respiratory syncytial virus by preventing viral attachment, internalization, and syncytium formation.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Blume is a popular traditional Chinese herbal medicine that has been used to manage respiratory tract disease, including common cold and chronic bronchitis for thousand years. Human respiratory syncytial virus (HRSV) is one of the leading causes of severe lower respiratory tract illness worldwide. No effective therapeutic modality against HRSV infection has been proved. It is unknown whether Cinnamomum cassia is effective against HRSV. AIM OF THE STUDY: This study tested the hypothesis that Cinnamomum cassia can effectively decrease HRSV-induced plaque formation and syncytium formation in respiratory mucosal cell lines. MATERIALS AND METHODS: Antiviral activity of the hot water extract of Cinnamomum cassia against HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Its ability to inhibit the synthesis of viral fusion (F) protein was examined by Western blot assay. RESULTS: Cinnamomum cassia dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (p<0.0001). Cinnamomum cassia was more effective when given before viral infection (p<0.0001) mainly by inhibition of viral attachment (p<0.0001) and internalization (p<0.0001). Cinnamomum cassia could inhibit F protein production and syncytium formation to interfere with HRSV spreading. CONCLUSIONS: Cinnamomum cassia prevented airway epithelia from HRSV infection through inhibiting viral attachment, internalization and syncytium formation. Cinnamomum cassia could be a candidate to develop therapeutic modalities to manage HRSV infection in the future.

Two novel fusion inhibitors of human respiratory syncytial virus.

To search for novel drugs against human respiratory syncytial virus (RSV), we have screened a diversity collection of 16,671 compounds for anti-RSV activity in cultures of HEp-2 cells. Two of the hit compounds, i.e., the N-(2-hydroxyethyl)-4-methoxy-N-methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthalazin-3-yl)benzenesulfonamide (designated as P13) and the 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane (designated as C15), reduced the virus infectivity with IC50 values of 0.11 and 0.13µM respectively. The concentration of P13 and C15 that reduced the viability of HEp-2 cells by 50% was 310 and 75µM respectively. Both P13 and C15 exhibited no direct virucidal activity or inhibitory effects on the virus attachment to cells. However, to inhibit formation of RSV-induced syncytial plaques P13 and C15 had to be present during the virus entry into the cells and the cell-to-cell transmission of the virus. The RSV multiplication in HEp-2 cells in the presence of P13 or C15 resulted in rapid selection of viral variants that were ∼1000 times less sensitive to these drugs than original virus. Sequencing of resistant viruses revealed presence of amino acid substitutions in the F protein of RSV, i.e., the D489G for C15-selected, and the T400I and N197T (some clones) for the P13-selected virus variants. In conclusion, we have identified two novel fusion inhibitors of RSV, and the detailed understanding of their mode of antiviral activity including selection for the drug resistant viral variants may help to develop selective and efficient anti-RSV drugs.

GI-5005, a yeast vector vaccine expressing an NS3-core fusion protein for chronic HCV infection.

The HCV nonstructural protein 3 (NS3) and core protein are highly conserved among various HCV genotypes, and several B- and T-cell epitopes have been characterized with these antigens. The immunotherapeutic vaccine GI-5005, being developed by GlobeImmune Inc, is a Tarmogen (targeted molecular immunogen) consisting of recombinant Saccharomyces cerevisiae yeast expressing an HCV NS3-core fusion protein designed to elicit antigen-specific host CD4+ and CD8+ T-cell responses for the treatment of chronic HCV infection. GI-5005 has demonstrated robust immunogenicity in preclinical in vitro and in vivo models. In a phase Ib clinical trial, GI-5005 monotherapy was well tolerated and displayed efficacy in patients with chronic HCV infection. At the time of publication, interim data were available from a completed phase II trial that evaluated a triple therapy of GI-5005 in combination with the standard-of-care (SOC; PEGylated-IFNalpha and ribavirin) regimen, compared with the SOC regimen alone. Triple therapy resulted in improved early virological responses in all treatment-naïve patients. End-of-trial results, including data of sustained virological responses, are required to better evaluate the efficacy of GI-5005 for the improvement of viral clearance and to compare the efficacy of the agent with other approaches such as NS3 protease inhibitors.

Influence of culture conditions on recombinant Drosophila melanogaster S2 cells producing rabies virus glycoprotein cultivated in serum-free medium.

The recombinant G glycoprotein from the surface of the rabies virus (RVGP) is a promising candidate as a rabies vaccine component and also for diagnostic purposes. In this study, RVGP production by transfected Drosophila melanogaster S2 cells cultivated in a serum-free medium (supplemented IPL-41 medium) was carried out. The effects of pH and pO(2) were evaluated in batch culture in parallel spinner flasks. The use of a pH equal to 6.3 and a pO(2) of 40% air saturation resulted in the highest RVGP content. These conditions were also used in fed-batch mode, yielding a RVGP content level of 98g/10(7) cells. The main nutrients consumed were glucose, glutamine, asparagine, serine and proline and the major metabolites produced were alanine and ammonia, according to the metabolism studies performed. Since RVGP is a transmembrane protein, two different methods for protein recovery were assessed and compared. Detergent-based cell disruption showed to be more effective than mechanical disruption with glass beads for glycoprotein recovery.

[Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand].

Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 100:1, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection.

A point mutation in the F1 subunit of human respiratory syncytial virus fusion glycoprotein blocks its cell surface transport at an early stage of the exocytic pathway.

Vaccinia virus recombinants expressing either wild-type or mutant forms of human respiratory syncytial (RS) virus (Long strain) fusion (F) glycoprotein were obtained. Proteolytic processing of the precursor, F0, and cell surface transport of the F glycoprotein were unaffected in the recombinants, except in those that contained the replacement Phe --> Ser at position 237 of the F1 subunit. In recombinants containing this mutation, either alone or in combination with others, the traffic of the F molecule was arrested at some intermediate step of its transport to the cell surface and, consequently, the endoproteolytic cleavage of the F0 precursor was inhibited. Immunofluorescence staining of infected cells and endoglycosidase H (Endo-H) sensitivity assays indicated that the arrest occurred before the mid-Golgi compartment. Dimerization and folding of the F protein were also affected by the Phe237 --> Ser substitution. Other amino acid replacements at positions 236 or 237 of the F1 subunit had various effects upon F0 maturation. These results are discussed in terms of the maturation requirements for the RS virus F molecule.

Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide".

Toward elucidating molecular details of virus-induced membrane fusion, we have studied the low pH-triggered interaction of the bromelain-solubilized ectodomain of influenza hemagglutinin with liposomes. Polypeptide segments which insert into the apolar phase of the lipid bilayer were first labeled specifically using either of the two membrane-restricted carbene-generating reagents, 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] undecanoyl]-sn-glycero-3-phosphorylcholine, and were then identified on the basis of cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine-skatole fragment analysis and Edman degradations. Here, we demonstrate that the hydrophobic interaction is mediated solely by the so-called "fusion peptide" which corresponds to the NH2-terminal segment of the BHA2 subunit of nature influenza hemagglutinin. Predominant sites of labeling within that segment were Phe-3, Ile-6, Phe-9, Trp-14, Met-17, and Trp-21. The average 3-4 residue spacing between consecutive labeled amino acid side chains suggests a helical structure of that segment with an amphiphilic character.