Synthesis of Novel Chiral Sulfonamide‐Bearing 1,2,4‐Triazole‐3‐thione Analogs Derived from D‐ and L‐Phenylalanine Esters as Potential Anti‐Influenza Agents

Novel enantiopure 1,2,4‐trizole‐3‐thiones containing a benzensulfonamide moiety were synthesized via multistep reaction sequence starting with D‐phenylalanine methyl ester and L‐phenylalanine ethyl ester as a source of chirality. The chemical structures of all compounds were characterized by elemental analysis, UV, IR, ¹H NMR, ¹³C NMR, 2D NMR (HETCOR), and mass spectral data. All compounds were tested in vitro antiviral activity against a broad variety of DNA and RNA viruses and in vitro cytostatic activity against murine leukemia (L1210), human T‐lymphocyte (CEM) and human cervix carcinoma (HeLa) cell lines. Although enantiopure 1,2,4‐triazole‐3‐thione analogs in (R) configuration emerged as promising anti‐influenza A H1N1 subtype in Madin Darby canine kidney cell cultures (MDCK), their enantiomers exhibited no activity. Especially compounds 18a , 21a , 22a , 23a , and 24a (EC₅₀: 6.5, 6.1, 2.4, 1.6, 1.7 μM, respectively) had excellent activity against influenza A H1N1 subtype compared to the reference drug ribavirin (EC₅₀: 8.0 μM). Several compounds have been found to inhibit proliferation of L1210, CEM and HeLa cell cultures with IC₅₀ in the 12–53 μM range. Compound 5a and 27a in (R) configuration were the most active compounds (IC₅₀: 12–22 μM for 5a and IC₅₀: 19–23 μM for 27a ). Chirality 28:495–513, 2016. © 2016 Wiley Periodicals, Inc.     

Direct inhibitory effect on viral entry of influenza A and SARS‐CoV‐2 viruses by azithromycin

ObjectivesUsing strategy of drug repurposing, antiviral agents against influenza A virus (IAV) and newly emerging SARS‐coronavirus 2 (SARS‐CoV‐2, also as 2019‐nCoV) could be quickly screened out.Materials and MethodsA previously reported engineered replication‐competent PR8 strain carrying luciferase reporter gene (IAV‐luc) and multiple pseudotyped IAV and SARS‐CoV‐2 virus was used. To specifically evaluate the pH change of vesicles containing IAV, we constructed an A549 cell line with endosomal and lysosomal expression of pHluorin2.ResultsHere, we identified azithromycin (AZ) as an effective inhibitor against multiple IAV and SARS‐CoV‐2 strains. We found that AZ treatment could potently inhibit IAV infection in vitro. Moreover, using pseudotyped virus model, AZ could also markedly block the entry of SARS‐CoV‐2 in HEK293T‐ACE2 and Caco2 cells. Mechanistic studies further revealed that such effect was independent of interferon signalling. AZ treatment neither impaired the binding and internalization of IAV virions, nor the viral replication, but rather inhibited the fusion between viral and vacuolar membranes. Using a NPC1‐pHluorin2 reporter cell line, we confirmed that AZ treatment could alkalize the vesicles containing IAV virions, thereby preventing pH‐dependent membrane fusion.ConclusionsOverall, our findings demonstrate that AZ can exert broad‐spectrum antiviral effects against IAV and SARS‐CoV‐2, and could be served as a potential clinical anti‐SARS‐CoV‐2 drug in emergency as well as a promising lead compound for the development of next‐generation anti‐IAV drugs.     

Membrane‐Based Approach for the Downstream Processing of Influenza Virus‐Like Particles

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration‐based approach is explored to purify influenza virus‐like particles. Switching from adsorptive separation to size‐based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities’ removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps.     

Increase of tumor necrosis factor‐α in the blood induces early activation of matrix metalloproteinase‐9 in the brain

Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy.     

ZstatFlu®‐II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics

The ZstatFlu®‐II test is a highly sensitive, specific, rapid, point‐of‐care chemiluminescent diagnostic test for influenza infection. Influenza viral neuraminidase‐specific substrate, spiroadamantyl‐1,2‐dioxetane‐4,7‐dimethoxy‐N‐acetyl‐neuraminic acid, is at the core of the ZstatFlu®‐II Test. The enzymatic reaction was carried out at 25°C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid? High Speed Detector Film. Positive results appeared as a ‘+’‐shaped white film image; negative results produced no image. The ‘glow’ kinetics, facilitated by a unique combination of light enhancers, also ‘tuned’ the wavelength of emission to match the spectral properties of the film. The substrate hydrolysed non‐enzymatically at acid pH or at temperatures above 25°C. In order to minimize false positives, the ZstatFlu®‐II Test was formatted with 0.3–0.4 K_m substrate and freezing the test kit until use. The pH optimization of the ZstatFlu®‐II test is discussed with reference to model compounds of sialyl‐glycosides. A nucleophilic attack or an electrostatic stabilization of a developing carbonium ion under the influence of the adjacent carboxyl group was probably responsible for non‐enzymatic hydrolysis of the substrate. Intramolecular general acid catalysis is proposed as a mechanism for the lability of the O‐glycosidic linkage of the substrate. Copyright © 2003 John Wiley & Sons, Ltd.     

Cell‐free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens

In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli‐based cell‐free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the “head” domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C‐terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines. Biotechnol. Bioeng. 2012; 109: 2962–2969. © 2012 Wiley Periodicals, Inc.     

Evaluation of influenza vaccine‐induced cell‐mediated immunity: Comparison between methods using peripheral blood mononuclear cells and whole blood

Assessment of cell‐mediated immunity (CMI) may be critical to evaluating the ability of individuals to protect themselves against influenza virus infection. However, it has been difficult to evaluate CMI because no simple means of measuring it is currently available. The aim of this study was to compare the performance of a CMI measurement method developed by us, which involves reacting whole blood with antigen, with the conventional method, which is based on isolating peripheral blood mononuclear cells (PBMCs). Correlations between these methods before and after vaccination of 26 healthy adults (aged 28–58 years; 12 men and 14 women) were assessed and changes in CMI after influenza vaccination in PBMCs cultured with antigen for 48 and 96 hr and whole blood cultured with antigen for 48 hr were studied. Results of CMI measurement using whole blood on Day 2 and PBMCs on Day 4 were found to be correlated. Spearman's correlation coefficients with four antigens (A [H1N1], A [H3N2], B [Yamagata lineage], and B [Victoria lineage]) before vaccination were 0.55, 0.61, 0.58, and 0.70, respectively and 0.40, 0.45, 0.62, and 0.52, respectively, after vaccination. CMI was detected sooner when whole blood was reacted with antigen than when PBMCs were reacted with antigen. The rate of positive reaction of influenza A (H1N1 and H3N2) in whole blood on Day 2 was higher than that in PBMCs on Day 2. Our method is simple and may be useful for vaccine development because it can measure CMI in a small amount of blood without separating off PBMCs.     

Novel Daidzein Analogs and Their in Vitro Anti‐Influenza Activities

A series of novel isoflavonoids were synthesized based on structural modifications of daidzein, an active ingredient of traditional Chinese medicine (TCM) and evaluated for their anti‐influenza activity, in vitro, against H1N1 Tamiflu‐resistant (H1N1 TR) virus in the MDCK cell line. Among them, 4‐oxo‐4H‐1‐benzopyran‐8‐carbaldehydes 11a – 11g were most promising, and they demonstrated better activities and selectivities comparable to those the reference ribarivin, a nucleoside antiviral agent. 3‐(4‐Bromophenyl)‐7‐hydroxy‐4‐oxo‐4H‐1‐benzopyran‐8‐carboxaldehyde ( 11c ) displayed the best inhibitory activity (EC₅₀, 29.0 μM ) and selectivity index (SI>10.3). Analysis of the structure? activity relationships (SAR) indicated that both the non‐naturally‐occurring Br‐substituted B‐ring and appropriate CHO and OH groups on the A‐ring might be critical for the activity and selectivity against H1N1 TR influenza viruses.     

Influence of a heptad repeat stutter on the pH‐dependent conformational behavior of the central coiled‐coil from influenza hemagglutinin HA2

The coiled‐coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled‐coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a “stutter,” a deviation of the idealized heptad repeat that is found in the central coiled‐coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter‐containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled‐coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH‐dependent coiled‐coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled‐coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH‐dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2. Proteins 2014; 82:2220–2228. © 2014 Wiley Periodicals, Inc.     

Real‐time monitoring of influenza virus production kinetics in HEK293 cell cultures

There is an increased interest from the vaccine industry to use mammalian cell cultures for influenza vaccine manufacturing. Therefore, it became important to study the influenza infection mechanism, the viral–host interaction, and the replication kinetics from a bioprocessing stand point to maximize the influenza viral production yield in cell culture. In the present work, influenza replication kinetics was studied in HEK293 cells. Two infection conditions were evaluated, a low (0.01) and a high multiplicity of infection (1.0). Critical time points of the viral production cycle (infection, protein synthesis, viral assembly and budding, viral release, and host‐cell death) were identified in small‐scale cell cultures. Additionally, cell growth, viability, and viral titers were monitored in the viral production process. The infection state of the cultivated cell population was assessed by influenza immunolabeling throughout the culture period. Influenza virus production kinetics were also on‐line monitored by dielectric spectroscopy and successfully correlated to real‐time capacitance measures. Overall, this work provided insights into the mechanisms associated with the infection of human HEK293 cell line by the influenza virus and demonstrated, once again, the usefulness of multifrequency scanning permittivity for in‐line monitoring and supervision of cell‐based viral production processes. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Insertion of single‐chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non‐recombinant Lactococcus lactis

Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N‐acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non‐recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single‐chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 ± 1.4% and 3,594.0 ± 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 ± 1.5% and 1,652.0 ± 34.1, respectively). Higher amount of HA1/L/AcmA (～2.9 × 10⁴ molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (～1.1 × 10⁴ molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154–162, 2017     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Synthesis and Olfactory Characterization of Silicon‐Containing Derivatives of the Acyclic Lily‐of‐the‐Valley Odorant 5,7,7‐Trimethyl‐4‐methylideneoctanal

5‐Methyl‐4‐methylidene‐6‐(trimethylsilyl)hexanal ( 1b ), a sila analog of the acyclic lily‐of‐the‐valley odorant 5,7,7‐trimethyl‐4‐methylideneoctanal ( 1a ), and the Si‐containing derivatives 2 – 6 were prepared in multistep syntheses, starting from Cl₃SiH and Cl₂SiMe₂, respectively. Compounds 1b, 2 – 6 , and their new precursors were characterized by elemental analyses (C, H, N) and NMR spectroscopic studies (¹H, ¹³C, ¹⁵N, and ²⁹Si). To gain more information about the structure? odor correlation in the family of lily‐of‐the‐valley or ‘muguet’ odorants, C/Si analogs 1a / 1b and derivatives 2 – 6 were evaluated for their olfactory properties.     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Homo‐C‐nucleoside analogs IV. Synthesis of 4‐(2,5‐anhydro‐D‐altro‐pentitol‐1‐yl)‐2‐phenyl‐2H‐1,2,3‐triazole homo‐C‐nucleoside. CD assignment of the absolute configuration of the carbon bridge

Dehydrative cyclization of 4‐(D‐altro ‐pentitol‐1‐yl)2‐phenyl‐2H ‐1,2,3‐triazole in basic medium with one moler equivalent of p‐toluene sulfonyl chloride in pyridine solution gave the homo‐C‐ nucleoside 4‐(2,5‐anhydro‐D‐altro ‐1‐yl)‐2‐phenyl‐2H ‐1,2,3‐triazole. The structure and anomeric configuration was determined by acylation, nuclear magnetic resonance (NMR), and mass spectroscopy. The stereochemistry at the carbon bridge of homo‐C‐ nucleoside 2‐phenyl‐2H ‐1,2,3‐triazoles was determined by circular dichroism (CD) spectroscopy.