Neuraminidase Inhibitor of Garcinia atroviridis L. Fruits and Leaves Using Partial Purification and Molecular Characterization

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. Garcinia atroviridis L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC₅₀ of 17.34–17.53 µg/mL or 91.22–92.21 µM against Clostridium perfringens-NA, and 56.71–57.85 µg/mL or 298.32–304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.     

Modelling the electrophoretic migration behaviour of peptides and glycopeptides from glycoprotein digests in capillary electrophoresis-mass spectrometry

In this study, the classical semiempirical relationships between the electrophoretic mobility and the charge-to-mass ratio (m_e vs. q/M^α) were used to model the migration behaviour of peptides and glycopeptides originated from the digestion of recombinant human erythropoietin (rhEPO), a biologically and therapeutically relevant glycoprotein. The Stoke’s law (α = 1/3), the classical polymer model (α = 1/2) and the Offord’s surface law (α = 2/3) were evaluated to predict migration of peptides and glycopeptides, with and without sialic acids (SiA), in rhEPO digested with trypsin and trypsin–neuraminidase. The Stoke’s law resulted in better correlations for the set of peptides used to evaluate the models, while glycopeptides fitted better with the classical polymer model. Once predicted migration times with both models, it was easy to simulate their separation electropherogram. Results were later validated predicting migration and simulating separation of a different set of rhEPO glycopeptides and also human transferrin (Tf) peptides and glycopeptides. The excellent agreement between the experimental and the simulated electropherograms with rhEPO and Tf digests confirmed the potential applicability of this simple strategy to predict, in general, the peptide–glycopeptide electrophoretic map of any digested glycoprotein.     

Multivalent neuraminidase hydrolysis resistant triazole-sialoside protein conjugates as influenza-adsorbents

Triazole-sialoside tailored proteins with high hemagglutinin (HA) and neuraminidase (NA) binding affinity are prepared. Dynamic light scattering shows that these pseudo-sialylated proteins are ideal virus capture macromolecules.     

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-β-D-glucoside, emodin-8-O-β-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.     

Neuraminidase inhibitory activity by compounds isolated from aerial parts of Rhinacanthus nasutus

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is known as traditional medicine for the treatment of various diseases, such as cancer, fungal infections, herpes virus infections and several types of skin diseases in South-East Asian countries. In this study, eight compounds 1–8 were isolated from the aerial parts of R. nasutus. The structures of compounds were determined by the spectroscopic methods, including 1D and 2D NMR. The isolated compounds were evaluated for neuraminidase inhibitory activity. Several lignans, 2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]-1,4-butanediol (5) and 8,8′-bisdihydrosiringenin glucoside (6), significantly inhibited neuraminidase activity, which was comparable to the positive controls, mangiferin and oseltamivir. In addition, a structure-based virtual screening against neuraminidase using bioactive components was demonstrated.     

A highly diastereoselective vinylogous Mannich condensation and 1,4-conjugate addition of (Z)-propenyl cuprate in the synthesis of an influenza neuraminidase inhibitor

A practical synthesis of neuraminidase influenza inhibitor, A-322278, has been developed. Asymmetry is introduced into the synthesis by an enzyme mediated ester hydrolysis. A highly diastereoselective vinylogous Mannich condensation reaction of N-Boc-2-tert-butyldimethylsilyloxypyrrole (TBSOP) and an N-(triphenylmethylsulfenyl)imine proceeds under thermodynamic control to assemble the framework. A significant temperature dependent rate difference for the transfer of (Z)- and (E)-propenyl moieties from a cuprate reagent during a 1,4-conjugate addition was observed. A very selective addition of cyanide to an N-acyliminium intermediate was employed to control the final stereocenter.     

A first synthesis of sulfonic acid analogues of N-acetylneuraminic acid

Sulfonic acid analogues of N-acetylneuraminic are synthesized from 1-thio-l-fucoside derivatives with the introduction of an azido group at C-4 of the fucose moiety and carbanionic addition onto fully protected lactones. The analogues in the form of methyl glycosides are subjected to a neuraminidase inhibition assay.     

Simultaneous stereoselective 4-amination with cyclic secondary amines and 2-O-deacetylation of peracetylated sialic acid derivatives

Treatment of methyl 5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-β-l-glycero-d-galacto-2-nonulopyranosidonate (1) with cyclic secondary amines in pyridine at room temperature for 24 h afforded unusual products (2a-g). Related experiments were carried out to explain the formation of 4-amination and 2-O-deacetylation of peracetylated sialic acid derivatives (2a-g). This reaction may provide a new strategy for the preparation of Zanamivir analogues as neuraminidase inhibitors for anti-H5N1 subtype of avian influenza virus (AIV).     

Macrocyclic Mechanism‐Based Inhibitor for Neuraminidases

A macrocyclic mechanism‐based inhibitor for neuraminidases (NAs) bearing a 2‐difluoromethylphenyl aglycone and a linker between the aglycone and C‐9 positions of sialic acid was synthesized and evaluated. The macrocyclic structure was designed to keep the aglycone moiety in the active site of the neuraminidase after cleavage of the glycoside bond. When Vibrio chorelae neuraminidase (VCNA) was treated with a similar acyclic derivative in the presence of detergent, the irreversible inhibition property was disabled. In contrast, this macrocyclic compound acted as an irreversible inhibitor for VCNA in the presence of detergent. Inhibition assay for various NAs using this macrocyclic compound revealed that the irreversible inhibition property depends on the k_cat of the neuraminidase treated. NAs having small k_cat values, such as Influenza viruses, Clostridium, Trypanosoma cruzi, and Human, were also inhibited irreversibly. However, Salmonella typhimurium NA, which has an extremely high k_cat, was not affected irreversibly by the inhibitor. Interestingly, in contrast to common k_cat inhibitors, the irreversibility of inhibition by this macrocyclic compound is inversely proportional to the k_cat of the target neuraminidase.     

Serendipitous Discovery of a Potent Influenza Virus A Neuraminidase Inhibitor

We have previously reported a potent neuraminidase inhibitor that comprises a carbocyclic analogue of zanamivir in which the hydrophilic glycerol side chain is replaced by the hydrophobic 3‐pentyloxy group of oseltamivir. This hybrid inhibitor showed excellent inhibitory properties in the neuraminidase inhibition assay (K_i=0.46 nM ; K_{i (zanamivir)}=0.16 nM ) and in the viral replication inhibition assay in cell culture at 10^?8 M . As part of this lead optimization, we now report a novel spirolactam that shows comparable inhibitory activity in the cell culture assay to that of our lead compound at 10^?7 M . The compound was discovered serendipitously during the attempted synthesis of the isothiourea derivative of the original candidate. The X‐ray crystal structure of the spirolactam in complex with the N8 subtype neuraminidase offers insight into the mode of inhibition.     

Novel Dual‐Site‐Binding Neuraminidase Inhibitor from Virtual Screening by Pharmacophore and Molecular Dynamics Methods

Neuraminidase is a significant anti‐influenza target that plays crucial role in virus replication cycle. The discovery of 150‐cavity in Group‐1 neuraminidase provides us a novel mentality of designing inhibitor which can bind with both conserved site and 150‐cavity. In order to discover novel dual‐site‐binding inhibitors, a 3D chemical‐feature‐based pharmacophore model was established to cover dual‐site in neuraminidase. The dual‐site‐binding model was consistent in predicting the binding conformation of Group‐1 neuraminidase inhibitor and applied for virtual screening of Specs database. Compound 4 (ZINC05790048) that aligned well to the model was selected after multiple filtrations for molecular dynamics simulations, indicating improved binding energy with neuraminidase. It can sever as the lead compound for a novel series of inhibitors.     

4-(N-乙酰氨基)-3-(4-芳基噻唑-2-氨基)苯甲酸乙酯的合成与生物活性

以3-氨基-4-乙酰氨基苯甲酸乙酯为原料设计合成了18种4-(N-乙酰氨基)-3-(4-芳基噻唑-2-基)-苯甲酸乙酯新化合物.化合物结构经质谱,元素分析,1H NMR和13C NMR确证.生物活性实验结果表明,化合物3d,3h,3p(40μg/mL)对神经氨酸酶(NA)的抑制率分别为36.02%,33.40%,42.05%;化合物3g,3h(500mg/L)对纹枯病菌的抑制率为50%.     

Enantioselective extraction of hydrophilic 2-chloromandelic acid enantiomers by hydroxypropyl-��-cyclodextrin: experiments and modeling

Enantioselective extraction of hydrophilic 2-chloromandelic acid (CMA) enantiomers from organic to aqueous phase with hydroxypropyl-??-cyclodextrin (HP-??-CD) as the selector was investigated. Equilibrium of the extraction system was modeled using a reactive extraction model with a homogeneous aqueous phase reaction. The influence of important process variables on the extraction efficiency, such as the type of the organic solvent and ??-cyclodextrin derivatives (??-CDs), concentration of the selector, pH and temperature, was investigated by experiment and modeling. Important parameters of this model were determined experimentally. Results showed that the experimental data agree with the model prediction perfectly and the model was further applied to accurately predict the extraction efficiency influenced simultaneously by pH and the concentration of HP-??-CD. Combining the experiment and the model data, the best extraction conditions were: pH of 2.5, HP-??-CD concentration of 0.05 mol L^?1, and temperature of 5°C, providing the enantioselectivity of 1.285 and the performance factor (pf) of 0.011.     

2,2′-Bipyridine-6,6′-diyl bisnitroxide as a paramagnetic host: Encapsulation of a zinc(II) ion

A chelate complex of zinc(II) and 2,2′-bipyridine-6,6′-diyl bis(tert-butyl nitroxide) (bpybNO) with a metal/ligand ratio of 1/2 was structurally characterized to be [Zn(bpybNO)₂][Zn(hfac)₃]₂, where Hhfac stands for 1,1,1,5,5,5-hexafluoropentane-2,4-dione. The magnetic susceptibility measurement indicates the presence of considerable antiferromagnetic interaction among the four S = 1/2 spins. The exchange parameter J was estimated as 2J/k_B = −103(1) K, on the basis of a tetrahedral coupling model. The antiferromagnetic coupling is stronger after complexation than before. The density-functional theory calculation on related model compounds supports the present analysis and clarified the role of the zinc ion as a superexchange coupler.     

Synthesis,biological activities and 3D-QSAR studies of (R)-2-phenyl-4,5-dihydrothiazole-4-carboxamide derivatives containing a sulfur ether moiety

A series of (R)-2-phenyl-4,5-dihydrothiazole-4-carboxamide derivatives containing a sulfur ether moiety were designed, synthesized, and tested for their antifungal activities against several phytopathogenic fungi. The established CoMSIA model could predict the antifungal activity.     

Production of octyl levulinate biolubricant over modified H-ZSM-5: Optimization by response surface methodology

The present study highlighted the use of modified H-ZSM-5 (Meso-HZ-5) as heterogeneous catalyst for the synthesis of octyl levulinate biolubricant by catalytic esterification of biomass derived renewable levulinic acid (LA) with n-octanol. The process variables such as catalyst loading (X₁), n-octanol to LA molar ratio (X₂) and reaction temperature (X₃) were optimized through response surface methodology (RSM), using Box-Behnken model. Analysis of variance was performed to determine the adequacy and significance of the quadratic model. The yield of octyl levulinate was obtained to be 99% at optimum process parameters. The developed quadratic model was found to be adequate and statistically accurate with correlation value (R²) of 0.9971 to predict the yield of octyl levulinate biolubricant. The study was also extended on the validation of theoretical and experimental data, including catalyst reusability.     

Liquid-liquid phase separation of binary Lennard-Jones fluid in slit nanopores

The capillary phase separation of a binary mixture of two truncated and shifted Lennard-Jones (LJ) Ar liquids in slit-shaped oxygen nanopores is examined. The LJ parameters—ε(Ar(A)–Ar(A))=ε(Ar(B)–Ar(B))=0.8ε(Ar(A)–Ar(B)) and 0.5ε(Ar(A)–O)?=?ε(Ar(B)–O)—were used to distinguish the two Ar liquids. The cut off distance for Ar was 3.5σ. We employed a molecular dynamics (MD) technique in which a pore space was connected with a bulk solution to easily determine the equilibrium bulk concentration. Liquid phase isotherms were obtained for pores with widths ranging from 5.5σ to 9.5σ, and the relation between the pore width and the phase separation concentration was determined. Each simulation was run until the bulk concentration attained equilibrium (1–2 μs). The MD results show that the Patrick model overestimates the bulk concentration for a given pore size. We proposed a modified Patrick model in which the pore wall potential is considered. In our model, the Gibbs-Tolman-Koenig-Buff effect is not considered for the interfacial tension since two surfaces of tension exist on both sides of the equimolar dividing surface of the two-Ar liquid phase. The two surfaces of tension neutralized Gibbs-Tolman-Koenig-Buff effect each other. The present simple model successfully describes the relation to prove its reliability.     

Estimation of stability constants of copper(II) and nickel(II) chelates with dipeptides by using topological indices

The estimation of the overall stability constants β₁ of copper(II) and nickel(II) chelates with dipeptides was performed by using the model with the valence connectivity index of the 3rd order (³χ^v). It was done by dividing β₁ to its constituting constants K₁ and K_a, and subsequently by dividing the basic set into subsets or treating the chelate rings as separate units. Altogether 15 copper(II) and eight nickel(II) chelates with dipeptides were investigated. Six models for the estimation of log K₁ and pK_a were checked, most of them by usage of indicator variables (for differentiation of copper(II) and nickel(II) complexes or subclasses of ligands). Estimates of log β₁ gave the range of rms values from 0.19 to 0.27. For the best model, errors of estimates were less than 0.34.     

The effect of the number of meaningful experimental data on the fitness of the theoretical copolymerisation models

Fifteen copolymers of maleic anhydride (MA or 0) and styrene (ST or 1) were prepared in a wide range of MA mole fraction in feed of f₀=0.01–0.90. ST (1) centred triad mole fractions (F₀₁₀, F_(011+110), F₁₁₁) were determined from quaternary ¹³C NMR spectra of aromatic “next to chain” carbon of ST units. Non-linear least squares (NLLS) curve fitting was used to fit the theoretical equations of the (1) terminal-, (2) penultimate unit effect-, (3) complex participation-, (4) complex dissociation- and (5) comppen-model to the experimental data of the triad mole fractions and the copolymer composition (Scheme. 1

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Scheme. 1. The propagating reactions and definitions of the reactivity ratios of the terminal-, penultimate unit effect-, complex participation-, complex dissociation- and comppen- copolymerisation models. 0 and 1 are the comonomers.

)>. The sum of squares (SS) and the reactivity ratios were calculated from these fitting processes. The smallest SS value of F₀₁₀ data, the test functions a and b and the “order grade matrix” consistently show that the (3) complex participation model is the most applicable model; while the (2) penultimate model is closely indicated for this copolymerisation. The (5) comppen model, unexpectedly, is the third best fit model. It can be explained by the small number of the meaningful experimental data (which is 10 for copolymer samples of f₀=0.01–0.40) compared to the number of variable parameters (p) of the comppen model (which is seven). It is suggested when the number of the meaningful experimental data (i.e. the triad fractions vary widely from 0 to 1) is much greater than the number of variable parameters (p) of the comppen model, the comppen model will be proved by the SS value to be the best fitted model in describing this MA–ST copolymerisation.     

Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter‐current chromatography

Toddalia asiatica (Linn.) Lam. is a medical plant traditionally used to treat coughs, fevers, and various diseases. Alkaloids are the main active ingredients in Toddalia asiatica (Linn.) Lam., but traditional methods for screening and separation are complex and labor‐intensive. In this work, an efficient strategy was developed to rapidly screen, identify, and separate neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. Ultrafiltration, high performance liquid chromatography, and time‐of‐flight mass spectrometry were employed for rapid screening and identification of neuraminidase inhibitors. A two‐phase solvent system comprising n‐hexane/ethyl acetate/methanol/water (5:5:3:7, v/v) was then selected for separation by high‐speed counter‐current chromatography. A sample loading of 200 mg and a stepwise flow rate were achieved by increasing the flow rate from 2 to 4 mL/min after 4 h. Three main fluoroquinoline alkaloids (haplopine, skimmianine, and 5‐methoxydictamnine) along with two coumarins were obtained via one‐step separation and their structures were determined by mass spectrometry and nuclear magnetic resonance. In vitro assays revealed skimmianine with half‐maximal inhibitory concentration of 16.2 ± 0.7 µmol/L was selected as the potential highest neuraminidase inhibitor. The results suggest that ultrafiltration high performance liquid chromatography–mass spectrometry combined with high‐speed counter‐current chromatography is efficient for the screening and isolation of neuraminidase inhibitors from complex natural products.