Jennifer L. McKimm-Breschkin

ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Detection of Influenza Viruses Resistant to Neuraminidase Inhibitors in Global Surveillance during the First 3 Years of Their Use

ABSTRACT Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) develops at a low level following drug treatment, and person-to-person transmission of resistant virus has not been recognized to date. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established to follow susceptibility of isolates and occurrence of NAI resistance at a population level in various parts of the world. Isolates from the WHO influenza collaborating centers were screened for susceptibilities to oseltamivir and zanamivir by a chemiluminescent enzyme inhibition assay, and those considered potentially resistant were analyzed by sequence analysis of the neuraminidase genes. During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, eight (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, one (0.22%) in 1999 to 2000, three (0.36%) in 2000 to 2001, and four (0.41%) in 2001 to 2002. Six had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use.

Resistance of influenza viruses to neuraminidase inhibitors: a review

Influenza virus is a negative stranded RNA virus. It contains two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA exists as a trimer and is responsible for binding to the terminal sialic acid bound to receptors on the surface of the target cell, leading to attachment and subsequent penetration by the virus into the cell. Influenza virus isolates from different animals appear to have a preference for specific receptor linkages. Equine and avian isolates bind preferentially to the a2,3 galactose structure, while human isolates bind preferentially to the a2,6 galactose structure (Leigh et al., 1995). A single amino acid mutation is sufficient to change receptor specificity (Rogers and Paulson, 1983; Nobusawa and Nakajima, 1988; Martin et al., 1998). Sequence analysis and alignment has identified key residues conserved across all HA subtypes, which are involved in receptor binding (Nobusawa et al., 1991). The sialic acid binding site forms a groove across the top of the HA surrounded by antibody binding sites (Weis et al., 1988). Residues 134–138 form the right side of the ligand binding site, and residues 224–228 form the left side. Other conserved residues appear to play a role in orienting several of the surface atoms for binding to the sialic acid, these include Tyr 98, Trp 153, His 183, Glu 190, Leu 194 and Tyr at 195 (Weis et al., 1988; Nobusawa et al., 1991). After replication of the virus, progeny virions bud from the cell surface. NA is thought to be responsible for cleavage of terminal sialic acid moieties from receptors, to facilitate elution of progeny virions from the infected cell. Since they are also glycosylated, newly synthesized HA and NA on virions may also contain sialic acid residues on their oligosaccharide chains. Removal of these terminal sugars is therefore also necessary to prevent self-aggregation, due to the HA of one virion binding to the sialic acids on an adjacent * Fax: +61-3-96627101. E-mail address: jennyb@bioresi.com.au (J.L. McKimmBreschkin).

Neuraminidase Sequence Analysis and Susceptibilities of Influenza Virus Clinical Isolates to Zanamivir and Oseltamivir

ABSTRACT The influenza virus neuraminidase (NA) inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world between 1999 and 2002. In order to monitor the potential development of resistance, the Neuraminidase Inhibitor Susceptibility Network was established to coordinate testing of clinical isolates collected through the World Health Organization influenza surveillance network from different regions of the world (M. Zambon and F. G. Hayden, Antivir. Res. 49:147-156, 2001). The present study establishes the baseline susceptibilities prior to and shortly after the introduction of the NA inhibitors. Over 1,000 clinical influenza isolates recovered from 1996 to 1999 were tested. Susceptibilities were determined by enzyme inhibition assays with chemiluminescent or fluorescent substrates with known NA inhibitor-resistant viruses as controls. The 50% inhibitory concentrations (IC50s) depended upon the assay method, the drug tested, and the influenza virus subtype. By both assays, the mean zanamivir IC50s were 0.76, 1.82, and 2.28 nM for the subtype H1N1 (N1), H3N2 (N2), and B NAs, respectively, and the oseltamivir IC50s were 1.2, 0.5, and 8.8 nM for the N1, N2, and B NAs, respectively. The drug susceptibilities of known zanamivir- and oseltamivir-resistant viruses with the NA mutations E119V, R292K, H274Y, and R152K fell well outside the 95% confidence limits of the IC50s for all natural isolates. Sequence analysis of the NAs of viruses for which the IC50s were above the 95% confidence limits and several control isolates for which the IC50s were in the normal range revealed variations in some previously conserved residues, including D151, A203, T225, and E375 (N2 numbering). Known resistance mutations are both influenza virus subtype and drug specific, but there was no evidence of naturally occurring resistance to either drug in any of the isolates.

Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase.

BACKGROUND Inhibitors of the influenza virus neuraminidase have been shown to be effective antiviral agents in humans. Several studies have reported the selection of novel influenza strains when the virus is cultured with neuraminidase inhibitors in vitro. These resistant viruses have mutations either in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic substituents have recently been reported and shown to select neuraminidase variants. This study seeks to clarify the structural and functional consequences of replacing the glycerol sidechain of the inhibitor with other chemical constituents. RESULTS The neuraminidase variant Arg292-->Lys is modified in one of three arginine residues that encircle the carboxylate group of the substrate. The structure of this variant in complex with the carboxamide inhibitor used for its selection, and with other Neu5Ac2en analogues, is reported here at high resolution. The structural consequences of the mutation correlate with altered inhibitory activity of the compounds compared with wild-type neuraminidase. CONCLUSIONS The Arg292-->Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292-->Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody

BACKGROUND While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.

Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance

Please cite this paper as: McKimm‐Breschkin (2012) Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza and Other Respiratory Viruses 7(Suppl. 1), 25–36.

Mechanism-Based Covalent Neuraminidase Inhibitors with Broad Spectrum Influenza Antiviral Activity

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71, published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially useful addition to the arsenal of antivirals used to treat influenza infection. Looking deeply into the mechanism of enzyme inhibition provides a clue for the development of new drugs to fight flu. Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Reduced Sensitivity of Influenza A (H5N1) to Oseltamivir

We tested the neuraminidase drug sensitivity of clade 1 and clade 2 influenza A virus (H5N1). All viruses demonstrated similar sensitivity to zanamivir, but compared to the 2004 clade 1 viruses, the Cambodian 2005 viruses were 6-fold less sensitive and the Indonesian clade 2 viruses were up to 30-fold less sensitive to oseltamivir.

Surveillance for neuraminidase-inhibitor-resistant influenza viruses in Japan, 1996-2007

BACKGROUND High usage of the neuraminidase inhibitor (NAI) oseltamivir in Japan since 2003 led the Neuraminidase Inhibitor Susceptibility Network to assess the susceptibility of community isolates of influenza viruses to oseltamivir and zanamivir. METHODS Isolates were tested by the enzyme inhibition assay and by neuraminidase (NA) sequence analysis. RESULTS Among 1,141 A(H3N2) viruses and 171 type B viruses collected in Japan during the 2003-2004 season, 3 (0.3%) A(H3N2) isolates showed high 50% inhibitory concentrations (IC(50)) to oseltamivir. Each possessed a known resistance NA mutation at R292K or E119V. During the 2004-2005 season, no resistance was found among 567 influenza A(H3N2) or 60 A(H1N1) isolates, but 1 of 58 influenza B isolates had an NAI resistance mutation (D197N). Sequence analysis found that 4 (3%) of 132 A(H1N1) viruses from 2005-2006 had known NA resistance mutations (all H274Y), but no additional resistant isolates were detected from that or the subsequent 2006-2007 season. Concurrent testing of a selection of 500 influenza B viruses from 2000 to 2006 showed significant variations between seasons in both oseltamivir and zanamivir IC(50) values, but no persistent increases over this period. CONCLUSIONS Our findings suggested possible low-level transmission of resistant variants from oseltamivir-treated patients in several seasons in Japan but no sustained reductions in NAI susceptibility or consistently increased frequency of detecting resistant variants for any strain or subtype, despite high levels of drug use. In particular, although oseltamivir-resistant A(H1N1) viruses with the H274Y mutation spread globally in 2007-2008, we found little evidence for increasing levels of resistant A(H1N1) variants in Japan in preceding years.