Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin

Abstract

Currently available drugs for the prevention and treatment of seasonal influenza virus infections are the M2 ion channel blockers (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (9). The clinical usefulness of amantadine and rimantadine is limited due to the increasing incidence of adamantane-resistant viruses in the population (3, 11). Moreover, the M2 ion channel blockers inhibit only influenza A virus replication and are associated with neurological side effects. NA inhibitors are favored clinically, since they are effective against all NA subtypes, are well tolerated, and have a higher barrier for resistance (27). However, drug-resistant isolates have been detected in A/H3N2-and A/H5N1-infected patients receiving oseltamivir treatment (10, 16). Even more reason for concern is the recent and worldwide isolation of oseltamivir-resistant A/H1N1 mutants, even among untreated patients (17, 46). Oseltamivir and, to a lesser extent, zanamivir have been stockpiled as part of pandemic preparedness plans and form the cornerstone of the response to the recent outbreak of the swine flu A/H1N1 virus (39, 40). However, it is unclear whether these antivirals will be sufficient to deal with larger influenza epidemics, so there is an urgent need to develop antivirals that act on a novel influenza virus target.An attractive antiviral strategy is to block influenza virus entry into the host cell, a process in which the viral hemagglutinin (HA) plays a key role (42). HA is a trimeric envelope glycoprotein that contains two disulfide-linked polypeptide chains, HA1 and HA2. After attachment of the receptor binding domain in the HA1 subunit to sialic acid-containing cell surface glycans, the virion is internalized by endocytosis. The acidic pH of the endosome leads to an extensive and irreversible conformational change of the HA protein, resulting in exposure of the fusion peptide, which inserts into the endosomal target membrane of the host cell (18). After fusion of the viral and endosomal membranes, the viral ribonucleoproteins are released into the cytosol and transported into the nucleus, where replication occurs (6). Crystallographic studies have provided detailed insight into the processes of HA refolding and extrusion of the fusion peptide (4). The latter is a sequence of hydrophobic amino acids located at the N terminus of the HA2 subunit, which is, in the prefusogenic conformation, sequestered in a pocket of ionizable residues at the monomer interface of the HA trimer (48). In order to exploit the HA protein as an antiviral target, several small-molecule inhibitors that block the acid-induced conformational change of HA have been identified (2,19,21,30,49). For many of these, development has been hindered by their subtype-dependent activities. On the other hand, these diverse fusion inhibitors represent excellent tools to identify the HA amino acid residues involved in the fusion process and/or delineate the structural differences among HA subtypes (36). We report here the i...

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

“…Protease sensitivity and Western blotting. The whole viral particles and purified HA proteins have successfully been used to demonstrate the acidic pH-induced HA conformational changes and HA sensitivity to proteolysis (15,38,(42)(43)(44)(45)(46). Following the acidic pH-induced conformational change of HA, L-(tosylamido-2-phenyl ethyl) chloromethyl ketone (TPCK)-treated trypsin digests the HA1 subunit but not the HA2 subunit (42).…”

Synthesis and biological evaluation of some new 1,3,4-thiadiazole and 1,2,4-triazole derivatives from L-methionine as antituberculosis and antiviral agents

“…The test compounds were evaluated for anti-influenza virus activity by a cytopathic effect (CPE) reduction assay (51,52). MDCK cells were infected with one of three influenza virus strains: A/H1N1, A/H3N2 and B.…”

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

“…influenza | neutralizing antibody | hemagglutinin | neutralization stoichiometry | membrane fusion R ecently described antibodies capable of neutralizing a broad range of influenza viruses through recognition of highly conserved epitopes on the hemagglutinin protein (HA) have potential use as antiinfluenza therapeutics and for rational design of vaccines, HA-binding proteins, and small molecules (1)(2)(3)(4)(5). Such diverse applications require a detailed understanding of the infection mechanisms that are disrupted by antibody binding.…”