a-315675 and Influenza--Human

Influenza is a major threat to millions of people worldwide. Vaccines and antiviral agents are two main options available to reduce the impact of the influenza virus, while anti-influenza agents are the most effective means to prevent the transmission of the highly contagious virus and to treat the epidemics of disease. At present, four anti-influenza agents have been approved by the FDA for the treatment of influenza, including two M2 protein ion channel inhibitors-amantadine and rimantadine and two neuraminidase inhibitors-zanamivir and oseltamivir. Arbidol hydrochloride, launched in Russia, is a potent inhibitor of influenza virus, too. Neuraminidase inhibitors could be classified generally by structure into six different kinds: sialic acid derivatives, benzoic acid derivatives, cyclohexene derivatives, cyclopentane derivatives, pyrrolidine derivatives and natural products. In this paper, recent progress in the research of the action mechanisms and structure-activity relationships of these anti-influenza virus agents were reviewed.

Topics: Acids, Carbocyclic; Amantadine; Antiviral Agents; Cyclopentanes; Guanidines; Humans; Indoles; Influenza, Human; Neuraminidase; Orthomyxoviridae; Oseltamivir; Pyrrolidines; Rimantadine; Structure-Activity Relationship; Viral Matrix Proteins; Zanamivir

Neuraminidase inhibitors (NAIs) play a key role in the management of influenza epidemics and pandemics. Given the novel pandemic influenza A(H1N1) (pH1N1) virus and the restricted number of approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority.. Recombinant pH1N1 viruses were generated by reverse genetics, expressing either the wild-type or any of 9 mutant neuraminidase (NA) proteins (N2 numbering: E119G, E119V, D198G, I222V, H274Y, N294S, S334N, I222V-H274Y, and H274Y-S334N). We evaluated these recombinant viruses for their resistance phenotype to 4 NAIs (oseltamivir, zanamivir, peramivir, and A-315675), NA enzymatic activity, and replicative capacity.. The E119G and E119V mutations conferred a multidrug resistance phenotype to many NAIs but severely compromised viral fitness. The oseltamivir- and peramivir-resistance phenotype was confirmed for the H274Y and N294S mutants, although both viruses remained susceptible to zanamivir. Remarkably, the I222V mutation had a synergistic effect on the oseltamivir- and peramivir-resistance phenotype of H274Y and compensated for reduced viral fitness, raising concerns about the potential emergence and dissemination of this double-mutant virus.. This study highlights the importance of continuous monitoring of antiviral drug resistance in clinical samples as well as the need to develop new agents and combination strategies.

Topics: Acids, Carbocyclic; Amino Acid Substitution; Antiviral Agents; Cyclopentanes; Drug Resistance, Viral; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Microbial Sensitivity Tests; Mutation, Missense; Neuraminidase; Oseltamivir; Pyrrolidines; Recombination, Genetic; Viral Plaque Assay; Viral Proteins; Virus Replication; Zanamivir

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (approximately 60-fold increase in its 50% inhibitory concentration [IC(50)] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (approximately 50- and 350-fold increases in IC(50), respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

Topics: Antiviral Agents; Cells, Cultured; Child, Preschool; Drug Resistance, Viral; Genetic Testing; Humans; Immunocompromised Host; Influenza A Virus, H3N2 Subtype; Influenza, Human; Male; Molecular Sequence Data; Neuraminidase; Oseltamivir; Point Mutation; Sequence Analysis, DNA

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC(50)s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC(50)s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC(50)s between the wild type and the variants, whereas the IC(50)s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.

Topics: Acids, Carbocyclic; Animals; Cell Line; Cyclopentanes; Dogs; Enzyme Assays; Enzyme Inhibitors; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Inhibitory Concentration 50; Neuraminidase; Oseltamivir; Zanamivir

Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010.. Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA.. With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility.. This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Topics: Acids, Carbocyclic; Adamantane; Amino Acid Substitution; Animals; Antiviral Agents; Cell Line; Cyclopentanes; Dogs; Drug Resistance, Viral; Enzyme Inhibitors; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Microbial Sensitivity Tests; Mutation, Missense; Neuraminidase; Oseltamivir; Pyrans; Pyrrolidines; Sialic Acids; Viral Plaque Assay; Zanamivir

Drug discovery efforts at Abbott Laboratories have led to the identification of influenza neuraminidase inhibitor A-315675 (1) as a candidate for development as an antiinfluenza drug. A convergent, stereoselective synthesis of this highly functionalized pyrrolidine is reported that utilizes pyrrolinone 2 as the key intermediate. The C5, C6 stereochemistry was established through a diastereoselective condensation of chiral imine compound 3 with silyloxypyrrole 4 to give pyrrolinone 2. The stereochemical outcome of this reaction depended critically on the choice of the imine functional group (FG), with tritylsulfenyl and (R)-toluenesulfinyl providing the desired products in good yields as crystalline intermediates. Conversion of pyrrolinone 2 into 1 was accomplished in seven subsequent steps, including Michael addition of cis-1-propenylcuprate at C4 and introduction of a cyano group as a carboxylic acid equivalent at C2.

Topics: Antiviral Agents; Humans; Influenza, Human; Magnetic Resonance Spectroscopy; Molecular Structure; Neuraminidase; Orthomyxoviridae; Pyrrolidines; Stereoisomerism