gs-4071 and peramivir

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.

Topics: Acids, Carbocyclic; Amino Acid Substitution; Animals; Antiviral Agents; Birds; Cyclopentanes; Dogs; Drug Resistance, Viral; Enzyme Inhibitors; Guanidines; Humans; Influenza in Birds; Influenza, Human; Madin Darby Canine Kidney Cells; Mutagenesis; Neuraminidase; Orthomyxoviridae; Oseltamivir; Reverse Genetics; Zanamivir

Peramivir is a potent neuraminidase (NA) inhibitor for treatment of influenza infection by intravenous administration. By replacing the carboxylate group in peramivir with a phosphonate group, phosphono-peramivir (6a), the dehydration and deoxy derivatives (7a and 8a) as well as their corresponding monoalkyl esters are prepared from a pivotal intermediate epoxide 12. Among these phosphonate compounds, the dehydration derivative 7a that has a relatively rigid cyclopentene core structure exhibits the strongest inhibitory activity (IC50 = 0.3-4.1 nM) against several NAs of wild-type human and avian influenza viruses (H1N1, H3N2, H5N1, and H7N9), although the phosphonate congener 6a is unexpectedly less active than peramivir. The inferior binding affinity of 6a is attributable to the deviated orientations of its phosphonic acid and 3-pentyl groups in the NA active site as inferred from the NMR, X-ray diffraction, and molecular modeling analyses. Compound 7a is active to the oseltamivir-resistant H275Y strains of H1N1 and H5N1 viruses (IC50 = 73-86 nM). The phosphonate monoalkyl esters (6b, 6c, 7b, 7c, 8b, and 8c) are better anti-influenza agents (EC50 = 19-89 nM) than their corresponding phosphonic acids (EC50 = 50-343 nM) in protection of cells from the viral infection. The phosphonate monoalkyl esters are stable in buffer solutions (pH 2.0-7.4) and rabbit serum; furthermore, the alkyl group is possibly tuned to attain the desired pharmacokinetic properties.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; Humans; Influenza, Human; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; Neuraminidase; Rabbits; Structure-Activity Relationship

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN-in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.

Topics: Acids, Carbocyclic; Antiviral Agents; Cities; Cyclopentanes; Drug Residues; Environmental Monitoring; Guanidines; Japan; Limit of Detection; Oseltamivir; Ozone; Pyrans; Risk Assessment; Rivers; Sewage; Sialic Acids; Water Pollutants, Chemical; Water Purification; Zanamivir

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme-inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.

Topics: Acids, Carbocyclic; Antiviral Agents; Buffers; Cyclopentanes; Drug Combinations; Drug Interactions; Drug Resistance, Viral; Enzyme Inhibitors; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Neuraminidase; Oseltamivir; Protein Binding; Solutions; Spectrometry, Fluorescence; Viral Proteins

Docking and molecular dynamics were used to study the nine ligands (see Scheme 1) at the neuraminidase (NA) active sites. Their binding modes are structurally and energetically different, with details given in the text. Compared with 1A (oseltamivir carboxylate), the changes of core template or/and functional groups in the other ligands cause the reductions of interaction energies and numbers of H-bonds with the NA proteins. Nonetheless, all these ligands occupy the proximity space at the NA active sites and share some commonness in their binding modes. The fragment approach was then used to analyze and understand the binding specificities of the nine ligands. The contributions of each core template and functional group were evaluated. It was found that the core templates rather than functional groups play a larger role during the binding processes; in addition, the binding qualities are determined by the synergistic effects of the core templates and functional groups. Among the nine ligands, 1A (oseltamivir carboxylate) has the largest synergistic energy and its functional groups fit perfectly with the NA active site, consistent with the largest interaction energy, numerous H-bonds with the NA active-site residues as well as experimentally lowest IC(50) value. Owing to the poorer metabolizability than oseltamivir, large contribution of the benzene core template and fine synergistic effects of the functional groups, the 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid should be an ideal lead compound for optimizing NA drugs.

Topics: Acids, Carbocyclic; Benzoates; Catalytic Domain; Cyclohexenes; Cyclopentanes; Drug Design; Guanidines; Humans; Hydrogen Bonding; Influenza, Human; Inhibitory Concentration 50; Ligands; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Neuraminidase; Orthomyxoviridae; Oseltamivir; Protein Binding; Static Electricity; Structure-Activity Relationship; Thermodynamics

Zanamivir and oseltamivir, specific inhibitors of influenza virus neuraminidase, have significantly different characteristics in resistance studies. In both cases resistance is known to arise through mutations in either the hemagglutinin or neuraminidase surface proteins. A new inhibitor under development by Biocryst Pharmaceuticals, BCX-1812, has both a guanidino group, as in zanamivir, and a bulky hydrophobic group, as in oseltamivir. Using influenza A/NWS/Tern/Australia/G70C/75 (H1N9), neuraminidase variants E119G and R292K have previously been selected by different inhibitors. The sensitivity of these variants to BCX-1812 has now been measured and found in both cases to be intermediate between those of zanamivir and oseltamivir. In addition, the X-ray crystal structures of the complexes of BCX-1812 with the wild type and the two mutant neuraminidases were determined. The ligand is bound in an identical manner in each structure, with a rearrangement of the side chain of E276 from its ligand-free position. A structural explanation of the mechanism of resistance of BCX-1812, relative to zanamivir and oseltamivir in particular, is provided.

Topics: Acetamides; Acids, Carbocyclic; Alphainfluenzavirus; Antiviral Agents; Crystallography, X-Ray; Cyclopentanes; Drug Resistance, Viral; Enzyme Inhibitors; Guanidines; Models, Molecular; Mutation; Neuraminidase; Oseltamivir; Protein Binding; Pyrans; Sialic Acids; Zanamivir

Topics: Acids, Carbocyclic; Administration, Oral; Animals; Antiviral Agents; Catalytic Domain; Crystallography, X-Ray; Cyclopentanes; Drug Design; Enzyme Inhibitors; Guanidines; Influenza A virus; Influenza B virus; Mice; Models, Molecular; Neuraminidase; Orthomyxoviridae Infections; Protein Binding; Stereoisomerism; Structure-Activity Relationship