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.     

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.     

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.     

Anti-neuraminidase activity of chemical constituents of Balanophora involucrata

Balanophora involucrata J. D. Hooker has been known to possess potential anti-inflammatory and antibacterial activities; however, its antiviral activity has not been evaluated so far. In order to find new neuraminidase inhibitors (NAIs), the neuraminidase (NA) inhibition activity of different B. involucrata extracts was evaluated. In this study, an in vitro NA inhibition assay was performed to identify which extract of B. involucrata exhibits (maximal) inhibitory activity against NA. Ultra high performance liquid chromatography/quadrupole time-of-flight–tandem mass spectroscopy (MS/MS) and molecular docking techniques were used to identify the specific compounds responsible for the anti-influenza activity of the extract, and to explore the potential natural NAIs. The ethyl acetate extract of B. involucrata exhibited significant inhibitory activity against NA with 50% inhibitory concentration (IC₅₀) value of 159.5 μg/mL. Twenty compounds were identified according to the MS/MS spectra; among them two compounds (quercitrin and phloridzin) showed obvious inhibitory activity against NA, with IC₅₀ of 311.76 and 347.32 μmol/L, respectively. This study suggested that B. involucrata can be a potential natural source of NAIs and may be useful in the fight against ferocious influenza viruses.     

三维全息原子场作用矢量(3D-HoVAIF)用于神经氨酸酶抑制剂的结构表征与设计及定量构效关系(QSAR)研究

对100个神经氨酸酶抑制剂抗禽流感药物结构并与其活性建立定量构效关系模型。采用本实验室提出的三维全息原子场作用矢量(3D-HoVAIF)对100个神经氨酸酶抑制剂进行结构表征,然后采用逐步回归对变量进行筛选后,运用偏最小二乘建立3D-HoVAIF描述子与神经氨酸酶抑制剂活性之间的QSAR模型。结果表明:复相关系数(R),交互校验的复相关系数(Q2)和模型的标准偏差(SD)分别为R2=0.805、Q2=0.657和SD=0.936,模型具有良好的稳定性和预测能力,并对文献中23个药物和设计的32个化合物进行了预测。表明三维全息原子场作用矢量能较好表征该类分子结构信息值得进一步推广应用。     

Inhibitory effect and possible mechanism of action of patchouli alcohol against influenza A (H2N2) virus

In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC?? of patchouli alcohol was above 20 μM. Patchouli alcohol could inhibit influenza virus with an IC?? of 4.03 ± 0.23 μM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of -40.38 kcal mol?1. The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.     

Exploring the Cause of Oseltamivir Resistance Against Mutant H274Y Neuraminidase by Molecular Simulation Approach

Oseltamivir (Tamiflu) is the preferred anti-viral drug employed to fight the flu virus in infected individuals. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infections. Until recently, only a low prevalence of neuraminidase inhibitors (NAIs) resistance (<1?%) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a H274Y neuraminidase mutation that was highly resistant to the NAI, oseltamivir. In this study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular docking and molecular dynamics simulation approach. Our results suggested that wild-type NA could be more indispensable for the oseltamivir binding, as characterized by minimum number of H-bonds, high flexibility and largest binding affinity than mutant-type NA. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.     

Assessing the performance of the g_mmpbsa tools to simulate the inhibition of oseltamivir to influenza virus neuraminidase by molecular mechanics Poisson–Boltzmann surface area methods

The molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method for GROMACS (g_mmpbsa) is an open-source tool that is capable of reading the trajectories generated by GROMACS and calculating the binding free energy using the MM-PBSA method. However, there are multiple force fields available for users to choose from in the GROMACS software, and there are also different solvent water models to combine with the chosen force fields. These different combinations of parameters may significantly impact the results of g_mmpbsa calculation. Unfortunately, the exact combination of force field and solvent water that can well calculate the free energy of the receptor–ligand binding in GROMACS has not been explored yet. To resolve the above issues, this study mainly explored the molecular dynamics (MD) simulations by GROMACS with the six commonly used force fields and three solvent water models, in combination with g_mmpbsa, to calculate the binding free energies of the influenza virus neuraminidase and its mutants with inhibitor oseltamivir carboxylate and compared the present results with previous published results of Amber software from ours and other researchers. Finally, we provided an optimized calculation model, as well as suggestions that may serve as advice and guidance for future computer-aided designs of drug molecules.     

Electrochemical Assay to Detect Influenza Viruses and Measure Drug Susceptibility

An electrochemical assay has been designed to rapidly diagnose influenza viruses. Exposure of a glucose‐bearing substrate to influenza viruses or its enzyme, neuraminidase (NA), releases glucose, which was detected amperometrically. Two methods were used to detect released glucose. First, we used a standard glucose blood meter to detect two viral NAs and three influenza strains. We also demonstrated drug susceptibility of two antivirals, Zanamivir and Oseltamivir, using the assay. Finally, we used disposable test strips to detect nineteen H1N1 and H3N2 influenza strains using this assay in one hour. The limit and range of detection of this first generation assay is 10² and 10²–10⁸ plaque forming units (pfu), respectively. Current user‐friendly glucose meters can be repurposed to detect influenza viruses.     

CrystalDock: a novel approach to fragment-based drug design

We present a novel algorithm called CrystalDock that analyzes a molecular pocket of interest and identifies potential binding fragments. The program first identifies groups of pocket-lining receptor residues (i.e., microenvironments) and then searches for geometrically similar microenvironments present in publically available databases of ligand-bound experimental structures. Germane fragments from the crystallographic or NMR ligands are subsequently placed within the novel binding pocket. These positioned fragments can be linked together to produce ligands that are likely to be potent; alternatively, they can be joined to an inhibitor with a known or suspected binding pose to potentially improve binding affinity. To demonstrate the utility of the algorithm, CrystalDock is used to analyze the principal binding pockets of influenza neuraminidase and Trypanosoma brucei RNA editing ligase 1, validated drug targets in the fight against pandemic influenza and African sleeping sickness, respectively. In both cases, CrystalDock suggests modifications to known inhibitors that may improve binding affinity.     

Rapid screening,identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC–ESI‐TOF‐MS combined with semipreparative HPLC

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.     

Isolation and structural identification of the main anthocyanin monomer in Vitis amurensis Rupr

Many types of anthocyanins are present in Vitis amurensis Rupr of ‘Beibinghong’, which is grown in North-east China and has high antioxidant activity. However, the anthocyanin with the highest content in V. amurensis Rupr has not yet been identified. In this study, pulsed electric field extraction and semi-preparative liquid phase separation were used to isolate the anthocyanin monomer from ‘Beibinghong’. UV–vis spectroscopy, FTIR spectroscopy, mass spectra and nuclear magnetic resonance were used to identify the anthocyanin monomer. The antioxidant activities of the anthocyanin monomer were also analysed. Malvidin-3,5-O-diglucoside was identified as the main anthocyanin in V. amurensis Rupr, which could be used as a raw material for its extraction. Furthermore, malvidin-3,5-O-diglucoside can be potentially used as a functional food, and a novel therapeutic and preventive agent for oxidative stress-related diseases. This study provides technical information for the future purification and structural identification of anthocyanins.     

Binding interaction analysis of the active site and its inhibitors for neuraminidase (N1 subtype) of human influenza virus by the integration of molecular docking, FMO calculation and 3D-QSAR CoMFA modeling

Recently, the worldwide spread of A/H5N1 avian influenza with high virulence has highlighted the potential threat of human influenza pandemic. Tamiflu and Relenza are currently the only two anti-influenza drugs targeting the neuraminidase (NA) enzyme of human influenza virus. Reports of the emergence of drug resistance further make the development of new potent anti-influenza inhibitors a priority. The X-ray crystallographic study of A/H5N1 avian influenza NA subtypes (Russell, R. J. Nature 2006, 443, 45-49) has demonstrated that there exist two genetically distinct groups, group-1 (N1, N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9), whose conformations are substantially different. The detailed comparison of their active sites has established, heretofore, the most accurate and solid molecular basis of structure and mechanism for the development of new anti-influenza drugs. In the present study, a three-dimensional structure of N1 subtype of human influenza type A virus (N1hA) has been generated by homology modeling using the X-ray crystallographic structure of N1 subtype of avian influenza virus (N1aA) as the template. Binding interaction analysis between the active site and its inhibitors has been performed by combining ab initio fragment molecular orbital (FMO) calculations and three-dimensional quantitative structure-activity relationship with comparative molecular field analysis (3D-QSAR CoMFA) modeling. Integrated with docking-based 3D-QSAR CoMFA modeling, molecular surface property (electrostatic and steric) mapping and FMO pair interaction analysis, a set of new receptor-ligand binding models and bioaffinity predictive models for rational design and virtual screening of more potent inhibitors of N1hA are established. In addition, the flexibility of the loop-150 of N1hA and N1aA has been examined by a series of molecular dynamics simulations.     

利用网络药理学方法研究热毒宁注射液抗流感病毒的分子作用机制

采用分子对接、网络分析预测热毒宁注射液抗流感病毒的分子作用机制, 并通过已建立的体外流感病毒神经氨酸酶筛选模型对网络预测结果验证. 结果表明, 热毒宁注射液所含化合物在化学空间上具有类药性质; 网络分析揭示出热毒宁注射液是通过与流感病毒吸附、脱壳、复制以及释放等环节的多个蛋白相互作用发挥抗流感病毒作用的; 对于预测的15 个活性分子而言, 实验结果初步证实, 对A型流感病毒, 木犀草素呈现较强的抑制作用, 槲皮素则呈现较弱的抑制作用, 这也初步证实了预测结果.     

Top leads for swine influenza A/H1N1 virus revealed by steered molecular dynamics approach

Since March 2009, the rapid spread of infection during the recent A/H1N1 swine flu pandemic has raised concerns of a far more dangerous outcome should this virus become resistant to current drug therapies. Currently oseltamivir (tamiflu) is intensively used for the treatment of influenza and is reported effective for 2009 A/H1N1 virus. However, as this virus is evolving fast, some drug-resistant strains are emerging. Therefore, it is critical to seek alternative treatments and identify roots of the drug resistance. In this paper, we use the steered molecular dynamics (SMD) approach to estimate the binding affinity of ligands to the glycoprotein neuraminidase. Our idea is based on the hypothesis that the larger is the force needed to unbind a ligand from a receptor the higher its binding affinity. Using all-atom models with Gromos force field 43a1 and explicit water, we have studied the binding ability of 32 ligands to glycoprotein neuraminidase from swine flu virus A/H1N1. The electrostatic interaction is shown to play a more important role in binding affinity than the van der Waals one. We have found that four ligands 141562, 5069, 46080, and 117079 from the NSC set are the most promising candidates to cope with this virus, while peramivir, oseltamivir, and zanamivir are ranked 8, 11, and 20. The observation that these four ligands are better than existing commercial drugs has been also confirmed by our results on the binding free energies obtained by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Our prediction may be useful for the therapeutic application.     

Structure-based virtual screening of influenza virus RNA polymerase inhibitors from natural compounds: Molecular dynamics simulation and MM-GBSA calculation

The resistances of matrix protein 2 (M2) protein inhibitors and neuraminidase inhibitors for influenza virus have attracted much attention and there is an urgent need for new drug. The antiviral drugs that selectively act on RNA polymerase are less prone to resistance and possess fewer side effects on the patient. Therefore, there is increased interest in screening compounds that can inhibit influenza virus RNA polymerase. Three natural compounds were found by using molecular docking-based virtual screening, which could bind tightly within the polymerase acidic protein-polymerase basic protein 1 (PA-PB1) subunit of influenza virus polymerase. Firstly, their drug likeness properties were evaluated, which showed that the hepatotoxicity values of all the three compounds indicating they had less or no hepatotoxicity, and did not have the plasma protein biding (PPB) ability, the three compounds needed to be modified in some aspects, like bulky molecular size. The stability of the complexes of PA-hits was validated through molecular dynamics (MD) simulation, revealing compound 2 could form more stable complex with PA subunit. The torsional conformations of each rotatable bond of the ligands in PA subunit were also monitored, to investigate variation in the ligand properties during the simulation, compound 3 had fewer rotatable bonds, indicating that the molecule had stronger rigidity. The bar charts of protein–ligand contacts and contacts over the course of trajectory showed that four key residues (Glu623, Lys643, Asn703 and Trp706) of PA subunit that participated in hydrogen-bond, water bridge and hydrophobic interactions with the hit compounds. Finally, the binding free energy and contributed energies were calculated by using MM-GBSA method. Out of the three compounds, compound 1 showed the lowest total binding free energy. Among all the interactions, the contribution of the covalent binding and the van der Waals energy were more than other items, compound 1 formed more stable hydrogen bonds with the residues of PA subunit binding pocket. This study smoothed the path for the development of novel lead compounds with improved binding properties, high drug likeness, and low toxicity to humans for the treatment of influenza, which provided a good basis for further research on novel and effective influenza virus PA-PB1 interaction inhibitors.     

Intermolecular Mechanism and Dynamic Investigation of Avian Influenza H7N9 Virus’ Susceptibility to E119V-Substituted Peramivir–Neuraminidase Complex

The H7N9 virus attaches itself to the human cell receptor protein containing the polysaccharide that terminates with sialic acid. The mutation of neuraminidase at residue E119 has been explored experimentally. However, there is no adequate information on the substitution with E119V in peramivir at the intermolecular level. Therefore, a good knowledge of the interatomic interactions is a prerequisite in understanding its transmission mode and subsequent effective inhibitions of the sialic acid receptor cleavage by neuraminidase. Herein, we investigated the mechanism and dynamism on the susceptibility of the E119V mutation on the peramivir–neuraminidase complex relative to the wildtype complex at the intermolecular level. This study aims to investigate the impact of the 119V substitution on the neuraminidase–peramivir complex and unveil the residues responsible for the complex conformations. We employed molecular dynamic (MD) simulations and extensive post-MD analyses in the study. These extensive computational investigations were carried out on the wildtype and the E119V mutant complex of the protein for holistic insights in unveiling the effects of this mutation on the binding affinity and the conformational terrain of peramivir–neuraminidase E119V mutation. The calculated total binding energy (ΔG_bind) for the peramivir wildtype is −49.09 ± 0.13 kcal/mol, while the E119V mutant is −58.55 ± 0.15 kcal/mol. The increase in binding energy (9.46 kcal/mol) is consistent with other post-MD analyses results, confirming that E119V substitution confers a higher degree of stability on the protein complex. This study promises to proffer contributory insight and additional knowledge that would enhance future drug designs and help in the fight targeted at controlling the avian influenza H7N9 virus. Therefore, we suggest that experimentalists collaborate with computational chemists for all investigations of this topic, as we have done in our previous studies.     

Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase‐imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus‐specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti‐neuraminidase antibodies.