Inhibition of Influenza A Virus Replication by Compounds Interfering with the Fusogenic Function of the Viral Hemagglutinin

Abstract

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.

Role of the viral hemagglutinin in the anti-influenza virus activity of newly synthesized polycyclic amine compounds

“…The HA 2 -F3L residue change selected under 6c was previously identified by Daniels et al (1985) in an Amtresistant avian A/H7N1 virus (Weybridge strain), which manifested an increase in hemolysis pH of 0.4 pH units. The HA 2 -F3L mutation was also reported by Plotch et al (1999), who selected a virus for resistance to the small molecule fusion inhibitor CL-61917, starting from an Amt-resistant A/FM/47 virus. The increased fusion pH of the HA 2 -F3L mutant virus is not unexpected, since this residue lies in the hydrophobic fusion peptide of HA.…”

Anti-influenza virus activity and structure–activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains

“…Although the HA has been extensively studied from a biochemical and epidemiological perspective, specific antiviral drugs blocking the HA-receptor interaction remain to be clinically developed. Several reports are available on the in vitro activity of small-molecule inhibitors of influenza virus fusion, which act by preventing the conformational change of the HA at low pH Deshpande et al, 2001;Plotch et al, 1999). Unfortunately, their development has been slow due to their inferior activity against some human influenza virus (sub)types, rapid selection for resistance and/or unsatisfactory outcome in animal models (Yagi et al, 1999).…”

Roles of the hemagglutinin of influenza A virus in viral entry and development of antiviral therapeutics and vaccines

“…By screening a chemical library, scientists at Wyeth-Ayerst Research have identified three compounds, CL61917 (an Nsubstituted piperidine), CL 385319 (the 5-fluoro analog of CL 61917), and CL62554 ( Fig. 5A), with inhibitory activity against H1 and H2 subtypes of IAVs with IC 50 at low micromolar levels (Plotch et al, 1999). Analysis of HA genes of the mutant viruses resistant to these compounds showed that single amino acid mutations clustered in the stem region of the HA trimer in and near the HA2 FP.…”

“…Within the endosomal compartment, the virion is exposed to the increasing acid pH 5-6, the HA protein undergoes an irreversible conformation change from its metastable prefusion conformation to a low-pH hairpin structure involving extrusion of the "fusion peptide (FP)" from the interior of the HA2 at the neutral-pH structure toward the endosomal membrane, promoting fusion of the viral and endosomal membranes (Harrison 2008;Reed et al, 2010). Xray crystallographic studies have demonstrated the extensive rearrangement of residues in HA2 at low pH with respect to their relative orientation and coil-coil formation, loop-to-helix or helix-to-loop transitions (Bullough et al, 1994;Durrer et al, 1996;Plotch et al, 1999;Harrison 2008).…”