Influenza Virus Neuraminidase Inhibitors Research Articles

Pharmacokinetic Modeling and Monte Carlo Simulation to Predict Interindividual Variability in Human Exposure to Oseltamivir and Its Active Metabolite, Ro 64-0802

Oseltamivir (Tamiflu®) is a prodrug of Ro 64-0802, a selective inhibitor of influenza virus neuraminidase. There is a possible relationship between oseltamivir treatment and neuropsychiatric adverse events; although this has not been established, close monitoring is recommended on the prescription label. The objective of this study was to predict interindividual variability of human exposure to oseltamivir and its active metabolite Ro 64-0802. By leveraging mathematical models and computations, physiological parameters in virtual subjects were generated with population means and coefficient of variations collected from the literature or produced experimentally. Postulated functional changes caused by genetic mutations in four key molecules, carboxylesterase 1A1, P-glycoprotein, organic anion transporter 3, and multidrug resistance-associated protein 4, were also taken into account. One hundred thousand virtual subjects were generated per simulation, which was iterated 20 times with different random number generator seeds. Even in the most exaggerated case, the systemic areas under the concentration–time curve (AUCs) of oseltamivir and Ro 64-0802 were increased by at most threefold compared with the population mean. By contrast, the brain AUCs of oseltamivir and Ro 64-0802 were increased up to about sevenfold and 40-fold, respectively, compared with the population means. This unexpectedly high exposure to oseltamivir or Ro 64-0802, which occurs extremely rarely, might trigger adverse central nervous system effects in the clinical setting.

Quantitative Structure -Activity Relations hip and Docking Studies on a Series of Substituted Acyl(thio)ureas and Thiadiazolo[2,3 - a a ] - pyrimidine Derivatives as Potent Inhibitors of Influenza Virus Neuraminidase

Quantitative Structure -Activity Relations hip and Docking Studies on a Series of Substituted Acyl(thio)ureas and Thiadiazolo[2,3 - a a ] - pyrimidine Derivatives as Potent Inhibitors of Influenza Virus Neuraminidase

Difluorosialic acids, potent novel influenza virus neuraminidase inhibitors, induce fewer drug resistance-associated neuraminidase mutations than does oseltamivir

Neuraminidase inhibitors (NAIs), including the most frequently prescribed oral therapeutic oseltamivir, play a critical role in the control of severe influenza virus (IFV) infections. However, recent reports of spread of an oseltamivir-resistant H1N1 pandemic strain in individuals who have never been exposed to oseltamivir highlight an urgent need for new antivirals against NAI-resistant IFVs. Difluorosialic acids (DFSAs) are a novel class of anti-IFV NAIs designed based on the mechanism of action of IFV NA, and distinguished by their covalent inhibition mode and their high structural similarity to the natural substrate, sialic acid. These characteristics should render the development of resistance a less rapid process. In this report, we evaluated the relative propensity of influenza A virus (IFV-A) NA to develop resistance against the DFSA class of inhibitor by passaging IFV-A strains in vitro in the presence of either oseltamivir or a representative DFSA (FeqGuDFSA). All the passage-selected lines gained mutations in hemagglutinin. Among the 12 oseltamivir-resistant passaged lines, five gained NA mutations and four of these were the well-defined H275Y mutation that causes oseltamivir resistance. In contrast, out of 15 DFSA-passaged lines, only 2 lines gained NA mutations. Further, NA inhibition assays indicated that these mutations did not change the sensitivity of NA to DFSA and thus the resistance to DFSA was not conferred by these NA mutations. These results strongly suggest that, compared to oseltamivir, IFV is less prone to development of resistance against DFSAs through NA mutations.

First I.V. neuraminidase inhibitorapproved to treat influenza

Abstract In December, FDA approved the first intravenously administered influenzavirus neuraminidase inhibitor, peramivir (Rapivab—BioCrystPharmaceuticals), for the treatment of acute uncomplicated influenza inadult patients who have not been symptomatic for more than 2 days.

Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection

The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8h shift. The most sensitive components of the test were the primers and probes targeting the A/H1pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction. LoDs for all targets in nasopharyngeal swab samples were ≤1000gc, with the exception of one target in the seasonal A/H1N1 subtype. Seasonal H275Y variants were detectable in a mixed population when present at >5% with wild type virus, while the 2009 pandemic H1N1 H275Y variant was detectable at ≤1% in a mixture with pandemic wild type virus. Influenza typing and subtyping results concurred with those obtained with real-time RT-PCR assays on more than 97% of the samples tested. The results demonstrate that a large panel of single-plex, real-time RT-PCR tests can be translated to an easy-to-use, sensitive, and specific microarray test for potential diagnostic use.

Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal

The influenza virus neuraminidase inhibitors are normally slow binding inhibitors, but many mutations leading to resistance, also result in the loss of the slow binding phenotype. Mutations can also affect the rate of dissociation of the inhibitors from the neuraminidase, but the assays to measure this require large amounts of virus and are time consuming. To more fully understand the impacts of mutations on the binding and dissociation of the neuraminidase inhibitors we have developed a solid phase reactivation assay, which can use small amounts of crude virus sample bound to an ELISA plate. Multiple viruses can be assayed simultaneously against multiple inhibitors. Using this assay we have demonstrated differences in the relative rates of dissociation of the inhibitors and reactivation of enzyme activity among different influenza A and B viruses for zanamivir, oseltamivir and peramivir. In general oseltamivir dissociated the fastest, and dissociation of peramivir was much slower than both the other inhibitors. Viruses with H274Y, E119V and E119G mutations demonstrated faster dissociation of the inhibitor to which they were resistant. Dissociation of zanamivir and oseltamivir were faster from the D197E mutant, but not of peramivir.

Identification of neuraminidase inhibitors by structure-based screening: promising new leads for influenza

Human influenza commonly known as seasonal flu which is caused by a RNA virus has been emerging as a major viral infection over the years. Virus neuraminidase inhibitors and M2 protein inhibitors are the agents which have been used to treat this viral infection. Among these two, viral neuraminidases named oseltamivir and zanamivir are most widely used as antiviral agents to treat influenza. But the recent emergence of resistance strains in the treatment with both zanamivir and oseltamivir creates a big problem to treat this viral infection effectively. In this study, we have designed 68 new human influenza virus neuraminidase inhibitors and reported them as new potential antiviral agents against the complex structure of influenza virus neuraminidase and sialic acid using various in silico tools and molecular docking analysis taking zanamivir as prototype.

A novel influenza virus neuraminidase inhibitor AV5027

A medium-sized focused library of novel Oseltamivir structural analogues with promising antiviral activity was successfully synthesized using a combinatorial approach. The synthesized compounds were then thoroughly evaluated in neuraminidase- and cell-based assays. As a result, (3R,4R,5S)-4-(2,2-difluoroacetylamino)-5-amino-3-(1-ethyl-propoxy)-cyclohex-1-enecarboxylic acid (AV5027) was identified as novel Hit-compound with picomolar potency. QSAR analysis was carried out based on the obtained biological data. Computational modeling was performed using a 3D-molecular docking approach and classical regression analysis. The developed integral model demonstrated a sufficient prediction accuracy and tolerance to evaluate compounds based on their potential activity against neuraminidase (NA) at least within the scaffold. Several compounds from the series can be reasonably regarded as promising anti-influenza drug-candidates.

Bioinspired total synthesis of katsumadain A by organocatalytic enantioselective 1,4-conjugate addition

Katsumadain A, a naturally occurring influenza virus neuraminidase (NA) inhibitor, was synthesized by using a bioinspired, organocatalytic enantioselective 1,4-conjugate addition of styryl-2-pyranone with cinnamaldehyde, followed by a tandem Horner–Wadsworth–Emmons/oxa Michael addition.

Neuraminidase inhibitor susceptibility profile of pandemic and seasonal influenza viruses during the 2009–2010 and 2010–2011 influenza seasons in Japan

Two new influenza virus neuraminidase inhibitors (NAIs), peramivir and laninamivir, were approved in 2010 which resulted to four NAIs that were used during the 2010–2011 influenza season in Japan. This study aims to monitor the susceptibility of influenza virus isolates in 2009–2010 and 2010–2011 influenza seasons in Japan to the four NAIs using the fluorescence-based 50% inhibitory concentration (IC50) method. Outliers were identified using box-and-whisker plot analysis and full NA gene sequencing was performed to determine the mutations that are associated with reduction of susceptibility to NAIs. A total of 117 influenza A(H1N1)pdm09, 59 A(H3N2), and 18 type B viruses were tested before NAI treatment and eight A(H1N1)pdm09 and 1 type B viruses were examined from patients after NAI treatment in the two seasons. NA inhibition assay showed type A influenza viruses were more susceptible to NAIs than type B viruses. The peramivir and laninamivir IC50 values of both type A and B viruses were significantly lower than the oseltamivir and zanamivir IC50 values. Among influenza A(H1N1)pdm09 viruses, the prevalence of H274Y viruses increased from 0% in the 2009–2010 season to 3% in the 2010–2011 season. These H274Y viruses were resistant to oseltamivir and peramivir with 200–300 fold increase in IC50 values but remained sensitive to zanamivir and laninamivir. Other mutations in NA, such as I222T and M241I were identified among the outliers. Among influenza A(H3N2) viruses, two outliers were identified with D151G and T148I mutations, which exhibited a reduction in susceptibility to oseltamivir and zanamivir, respectively. Among type B viruses, no outliers were identified to the four NAIs. For paired samples that were collected before and after drug treatment, three (3/11; 27.3%) H274Y viruses were identified among A(H1N1)pdm09 viruses after oseltamivir treatment but no outliers were found in the laninamivir-treatment group (n=3). Despite widespread use of NAIs in Japan, the prevalence of NAI-resistant influenza viruses is still low.

Construction of a novel system for developing inhibitors of influenza virus neuraminidase by yeast cell surface engineering

Neuraminidase (NA) is a glycoprotein of the influenza virus membrane and is involved in releasing newly made virus from infected cells, and thus it is the main target of the current influenza chemotherapy. Novel inhibitors of NA which is easily mutated are urgently required to be developed because of the recent emergence of oseltamivir‐resistant strains and highly pathogenic avian influenza viruses. Yeast cell surface engineering is one of the useful technologies for screening binding proteins with specific affinity to target molecules. In this study, we attempted to construct the efficient system for developing inhibitors of NA using this technology. The head domain of wild‐type or oseltamivir‐resistant (H275Y) H5N1 NA was displayed on yeast cell surface and their activities were evaluated. These results suggested that the displayed head domain of NAs retained the innate characteristics of NAs and was useful for evaluating inhibition activity[1].

Trapping influenza neuraminidase

A team from The University of British Columbia has used a mechanism-based approach to design irreversible inhibitors of influenza virus neuraminidase. The compounds are being developed by the venture arm of The Centre for Drug Research and Development, a public-private partnership that commercializes discoveries from Canadian researchers.

Molecular docking, molecular dynamics simulation, and QSAR model on potent thiazolidine-4-carboxylic acid inhibitors of influenza neuraminidase

In the current work, the relationship between the structure and activity of a series of novel thiazolidine-4-carboxylic acid derivatives as potent influenza virus neuraminidase inhibitors was studied using docking, molecular dynamics (MD) simulations, and QSAR analysis. A 7,000 ps MD simulation in a cubic water box were employed to build 3D structure of the 2HU4 in a water environment. After reaching the equilibrium, the inhibitors were docked into the 2HU4 to realize the binding site of the enzyme. The docking analysis showed that the interaction of the inhibitors with residues Arg371, Arg430, Gly429, Ile427, Lys432, Pro431, Trp403, and Tyr347 plays an important role in the activities of the inhibitors. The docked configurations of the inhibitors with the lowest free energy were used to calculate the most feasible descriptors. The selected descriptors were related to the inhibitory activities using stepwise multiple linear regression, classification and regression trees, and least squares support vector regression techniques. The satisfactory results (R p 2 = 0.883, Q LOO 2 = 0.872, R L25%O 2 = 0.835, RMSELOO = 0.310, and RMSEL25%O = 0.352) demonstrate that CART-LS-SVR models present the relationship between influenza virus neuraminidase inhibitors activity and molecular descriptors clearly. An energetic analysis based on MD calculations, revealed that the potency of the most active compound binding is governed by electrostatic and van der Waals contacts. The results provide a set of useful guidelines for the rational design of novel influenza virus neuraminidase inhibitors.

Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: The hydrophobic side chain influences type A subtype selectivity

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in order to investigate structure–activity relationships. The results indicated that size and geometry of the C3-side chain are important for selectivity of inhibition against N1 versus N2 NA, important type A influenza variants that infect man, including the highly lethal avian influenza.

Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice

Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5d starting 4h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100mg/kg/d. Peramivir was 30% protective at 0.5mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10–50% when the 0.025, 0.05, and 0.1mg/kg/d doses of peramivir were combined with 20mg/kg/d favipiravir and when all doses of peramivir were combined with 40mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1mg/kg/d doses of peramivir combined with favipiravir at the 20mg dose level. Therefore, the 20mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5mg/kg/d (1mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.

Studies on Synthesis and Structure-Activity Relationship (SAR) of Derivatives of a New Natural Product from Marine Fungi as Inhibitors of Influenza Virus Neuraminidase

Based on the natural isoprenyl phenyl ether from a mangrove-derived fungus, 32 analogues were synthesized and evaluated for inhibitory activity against influenza H1N1 neuraminidase. Compound 15 (3-(allyloxy)-4-hydroxybenzaldehyde) exhibited the most potent inhibitory activity, with IC50 values of 26.96 μM for A/GuangdongSB/01/2009 (H1N1), 27.73 μM for A/Guangdong/03/2009 (H1N1), and 25.13 μM for A/Guangdong/ 05/2009 (H1N1), respectively, which is stronger than the benzoic acid derivatives (~mM level). These are a new kind of non-nitrogenous aromatic ether Neuraminidase (NA) inhibitors. Their structures are simple and the synthesis routes are not complex. The structure-activity relationship (SAR) analysis revealed that the aryl aldehyde and unsubstituted hydroxyl were important to NA inhibitory activities. Molecular docking studies were carried out to explain the SAR of the compounds, and provided valuable information for further structure modification.

Effect of Milk on the Pharmacokinetics of Oseltamivir in Healthy Volunteers

We previously showed that oseltamivir, a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802, is a substrate of proton-coupled oligopeptide transporter (PEPT1), and its intestinal absorption in rats is markedly inhibited by administration with milk. To investigate the importance of PEPT1 for oseltamivir absorption in humans, and the characteristics of the drug–milk interaction, a crossover clinical study was conducted in healthy volunteers, who received 75mg of oseltamivir with 400mL of water or milk. Milk significantly reduced the maximum plasma concentration (Cmax) and the area under the plasma concentration–time curve from 0 to 2h (AUC0–2) of both oseltamivir and Ro 64-0802 (oseltamivir, 68.9% and 34.5%; Ro 64-0802, 69.5% and 14.2%, respectively, vs. water), but had no significant effect on the apparent terminal half-life (t1/2) or AUC0–∞. Urinary recovery of oseltamivir and Ro 64-0802 was significantly reduced to 77.5% of the control by milk. The early reduction of oseltamivir absorption might be through the PEPT1 inhibition by milk peptides. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the PEPT1 expression and its contribution. This might be the first report suggesting the clinical drug–food interaction via PEPT1. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3854–3861, 2011

Characteristic of alkylated chalcones from Angelica keiskei on influenza virus neuraminidase inhibition

As part of our ongoing effort to develop influenza virus neuraminidase (NA) inhibitors from various medicinal plants, we utilized bioassay-guided fractionation to isolated six alkylated chalcones (1-6) from Angelica keiskei. Xanthokeistal A (1) emerged as new compound containing the rare alkyl substitution, 6,6-dimethoxy-3-methylhex-2-enyl. When we tested the ability of these individual alkyl substituted chalcones to inhibit influenza virus NA hydrolysis, we found that 2-hydroxy-3-methyl-3-butenyl alkyl (HMB) substituted chalcone (3, IC(50)=12.3 μM) showed most potent inhibitory activity. The order of potency of substituted alkyl groups on for NA inhibition was HMB>6-hydroxyl-3,7-dimethyl-octa-2,7-dienyl>dimethylallyl>geranyl. All NA inhibitors screened were found to be reversible noncompetitive inhibitors.

Molecular Docking and QSAR Studies on Substituted Acyl(thio)urea and Thiadiazolo [2,3‐α] Pyrimidine Derivatives as Potent Inhibitors of Influenza Virus Neuraminidase

Surflex-Dock was employed to dock 36 thiourea and thiadiazolo [2,3-alpha] pyrimidine derivatives into neuraminidase 1a4g. Molecular docking results showed that hydrogen bonding, electrostatic, and hydrophobic features were important factors affecting inhibitory activities of these neuraminidase inhibitors. Moreover, there was a significant correlation between the predicted binding affinity (total scores) and experimental pIC50 values with correlation coefficient r=0.846 and p<0.0001. Hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis were used to develop quantitative structure-activity relationship models. Squared multiple correlation coefficients (r2) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.899, 0.878, and 0.865, respectively. Squared cross-validated correlation coefficient (q2) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models was in turn 0.628, 0.656, and 0.509. In addition, squared multiple correlation coefficients for test set (r2test) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.558, 0.667, and 0.566, respectively. The most active sample ID 2 was taken as a template molecule to design new molecules. Based on the comparative molecular field analysis model, new compounds were designed by LeapFrog. Seven new compounds with improved binding energy and predicted activities were finally obtained.

Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32–100 μM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01–10 nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations. Infected mice were treated twice daily for 5 days starting 2 h prior to virus challenge using drug doses of 0.05–0.4 mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4 mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2 mg/kg/day) compared to single drug treatments. The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.