gs-4071 and Orthomyxoviridae-Infections

Since the neuraminidase (NA) enzyme of the influenza A virus plays a key role in the process of release of new viral particles from a host cell, it is often a target for new drug design. The emergence of NA mutations, such as H275Y, has led to great resistance against neuraminidase inhibitors, including oseltamivir and zanamivir. Hence, we herein designed a set of derivatives by modifying the amine and/or carboxylic groups of oseltamivir. After being screened for their physicochemical (Lipinski's rule) and toxicological properties, the remaining compounds were submitted to molecular and theoretical studies. The docking simulations provided insights into NA recognition patterns, demonstrating that oseltamivir modified at the carboxylic moiety and coupled with anilines had higher affinity and a better binding pose for NA than the derivatives modified at the amine group. Based on these theoretical studies, the new oseltamivir derivatives may have higher affinity to mutant variants and possibly to other viral subtypes. Accordingly, two compounds were selected for synthesis, which together with their respective intermediates were evaluated for their cytotoxicity and antiviral activities. Their biological activity was then tested in cells infected with the A/Puerto Rico/916/34 (H1N1) influenza virus, and virus yield reduction assays were performed. Additionally, by measuring neuraminidase activity with the neuraminidase assay kit it was found that the compounds produced inhibitory activity on this enzyme. Finally, the infected cells were analysed with atomic force microscopy (AFM), observing morphological changes strongly suggesting that these compounds interfered with cellular release of viral particles.

Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Computer Simulation; Dogs; Drug Design; Drug Resistance, Viral; Enzyme Inhibitors; HeLa Cells; Humans; In Vitro Techniques; Influenza A Virus, H1N1 Subtype; Influenza, Human; Madin Darby Canine Kidney Cells; Microscopy, Atomic Force; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Vero Cells; Viral Proteins

A target-free and cell-based approach was applied to evaluate the anti-influenza properties of six newly synthesized benzoic acid derivatives. SA-2, the ethyl 4-(2-hydroxymethyl-5-oxopyrrolidin-1-yl)-3-[3-(3-methylbenzoyl)-thioureido] benzoate (compound 2) was screened as a potential drug candidate. In a cytopathic effect assay, SA-2 dose dependently inhibited H1N1, H3N2 and the oseltamivir-resistant mutant H1N1-H275Y influenza viruses in both virus-infected MDCK and A549 cells, with 50% effective concentrations (EC50) in MDCK cells of 9.6, 19.2 and 19.8 μM respectively, and 50% cytotoxic concentration (CC50) of 444.5 μM, showing competitive antiviral activity with oseltamivir in vitro. Orally administered SA-2 effectively protected mice infected with lethal doses of H1N1 or oseltamivir-resistant strain H1N1-H275Y, conferring 70% or 50% survival at a dosage of 100 mg/kg/d, reducing body weight loss, alleviating the influenza-induced acute lung injury, and reducing lung virus titer. Mechanistic studies showed that SA-2 efficiently inhibited the activity of RNA polymerase and suppressed NP and M1 levels during viral biosynthesis by interfering with gene transcription without having an obvious influence on virus entry and release. Based on these favourable findings, SA-2, a novel anti-influenza agent, with its potent anti-influenza activity in vitro and in vivo, could be a promising antiviral for the treatment of infection of influenza A viruses, including oseltamivir-resistant mutants.

Topics: A549 Cells; Acute Lung Injury; Animals; Antiviral Agents; Benzoates; DNA-Directed RNA Polymerases; Dogs; Drug Evaluation, Preclinical; Drug Resistance, Viral; HEK293 Cells; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza, Human; Madin Darby Canine Kidney Cells; Male; Mice; Mice, Inbred ICR; Orthomyxoviridae Infections; Oseltamivir; Ribavirin; Virus Replication

Oseltamivir phosphonic acid (tamiphosphor, 3a), its monoethyl ester (3c), guanidino-tamiphosphor (4a), and its monoethyl ester (4c) are potent inhibitors of influenza neuraminidases. They inhibit the replication of influenza viruses, including the oseltamivir-resistant H275Y strain, at low nanomolar to picomolar levels, and significantly protect mice from infection with lethal doses of influenza viruses when orally administered with 1 mg/kg or higher doses. These compounds are stable in simulated gastric fluid, liver microsomes, and human blood and are largely free from binding to plasma proteins. Pharmacokinetic properties of these inhibitors are thoroughly studied in dogs, rats, and mice. The absolute oral bioavailability of these compounds was lower than 12%. No conversion of monoester 4c to phosphonic acid 4a was observed in rats after intravenous administration, but partial conversion of 4c was observed with oral administration. Advanced formulation may be investigated to develop these new anti-influenza agents for better therapeutic use.

Topics: Acetamides; Administration, Oral; Alphainfluenzavirus; Animals; Antiviral Agents; Betainfluenzavirus; Biological Availability; Blood Proteins; Cyclohexenes; Cytopathogenic Effect, Viral; Dogs; Drug Resistance, Viral; Drug Stability; Female; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Madin Darby Canine Kidney Cells; Male; Mice; Mice, Inbred BALB C; Microsomes, Liver; Mutation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Phosphorous Acids; Protein Binding; Rats; Structure-Activity Relationship

Pandemic 2009 influenza A (H1N1) virus (H1N1pdm) is different from contemporary seasonal human viruses in that it can cause infection deep in the lungs of critical care patients. Here we establish a mammalian animal model and assessed the efficacy of the neuraminidase (NA) inhibitor oseltamivir treatment against H1N1pdm virus infection. Oseltamivir (25 mg/kg/day twice daily for 5 days) was orally administered to groups of ferrets, starting either 2 or 24 h after inoculation with 10(6)PFU of A/California/04/2009 (H1N1) influenza virus. We determined that virus replication was restricted to 1 or 2 of 4 lung lobes in oseltamivir-treated animals, while virus was consistently isolated from 4 of 4 lung lobes in control animals (1.5-3.8log(10)PFU/g). Analysis of arterial blood oxygenation revealed less pronounced changes in partial oxygen and carbon dioxide pressure in oseltamivir-treated ferrets, and histologic examination confirmed reduced pneumonia. Treated animals had significantly decreased inflammatory responses in the upper respiratory tract (P < 0.05), less fever and weight loss, and less reduction of activity. Virus titers in the nasal washes of treated and control ferrets did not differ significantly. NA sequencing and fluorescence-based phenotypic assays identified no oseltamivir-resistant variants. Overall, oseltamivir treatment decreases the signs of infection and reduced the spread of H1N1pdm influenza virus in the lungs of ferrets and therefore impeded the development of viral pneumonia.

Topics: Administration, Oral; Animals; Antiviral Agents; Cell Line; Disease Models, Animal; Dogs; Drug Resistance, Viral; Female; Ferrets; Influenza A Virus, H1N1 Subtype; Lung; Microbial Sensitivity Tests; Mutation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Oxygen; Pneumonia, Viral; Viral Load; Viral Plaque Assay; Virus Replication

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.

Topics: Animals; Antiviral Agents; Cell Line; Crystallography; Disease Models, Animal; Dogs; Drug Resistance, Viral; Enzyme Inhibitors; Ferrets; Humans; Influenza A Virus, H5N1 Subtype; Kidney; Male; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Protein Structure, Tertiary; Recombinant Proteins; 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