Novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin

Abstract

A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 muM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.

FIG. 1.

Strategy for chemical synthesis of the compounds.

FIG. 2.

Inhibitory effect of 4c on vRNA synthesis as a function of the time of compound addition. MDCK cells were infected with A/X-31 influenza virus, and compounds were added at −30 min, 0 h, 30 min, 1 h, 3 h, 5 h, or 8 h p.i. At 10 h p.i., the cells were subjected to RNA extraction for subsequent analysis by real-time RT-PCR. The vRNA synthesis is presented as the fold increase in vRNA copies at 10 h p.i. relative to the number of added virus particles. The data shown are the means ± standard deviations (SD) (the numbers of experiments are in parentheses).

FIG. 3.

4c inhibits low-pH-induced polykaryon formation. HeLa cells expressing wild-type A/X-31 HA were treated with trypsin to cleave HA0, washed, and incubated in the presence of various concentrations of 4c. Then, the pH was lowered to 5.0, and the cells were incubated for 15 min at 37°C in the presence of 4c. Following syncytium formation for 3 h at 37°C in medium, the cells were fixed, stained with Giemsa, and examined by microscopy (original magnification, ×200). Representative fields are shown.

FIG. 4.

Effect of 4c on the conformational change of HA, as demonstrated by trypsin digestion. A/X-31 virus was incubated at 37°C for 15 min in the presence of various concentrations of 4c, and the pH was lowered to 5.0. After neutralization, the mixtures were treated with trypsin. The lysates were subjected to Western blot analysis under reducing conditions and using an anti-HA1 antibody. The low-pH-induced conformational change of the HA resulted in the disappearance of the HA1 band.

FIG. 5.

pH dependence of hemolysis for wild-type and 4c-resistant mutants. A hemolysis assay was performed in which the pH of the acidic buffer was varied between 4.6 and 6.0. The y axis shows the degree of hemolysis, expressed as the optical density at 450 nm (OD₅₄₀) minus the blank value obtained from a mock-infected condition. 4c^r-A, 4c^r-B, and 4c^r-C, clones selected under 4c pressure; control, pooled data from three clones selected in the absence of compound. The values in parentheses are the pHs at which 50% hemolysis occurred relative to the value at pH 4.9.

FIG. 6.

Positions of 4c-associated HA mutations and the predicted binding pocket of 4c in the HA protein. The modeling was based on the published crystal structure of the X-31 HA in complex with TBHQ (36). (A) Ribbon diagram showing the locations of the amino acid residues (R220₁, A43₂, E57₂, A96₂, and D112₂) involved in 4c resistance. The HA1 chains are colored in sky blue (A chain), royal blue (C chain), and slate blue (E chain); HA2 chains are in pink (B chain), flesh (D chain), and purple (F chain); and the fusion peptide is indicated in green. The frame indicates the position of the binding pocket of 4c. (B) Chimera model of the interactions of 4c in the binding pocket around glutamic acid-57. The numbering of the amino acid residues includes the polypeptide chain (HA1 chains, A, not visible; C, white; and E, cyan; HA2 chains, B, not visible; D, orange; and F, khaki). Predicted hydrogen bonds with the main chain carbonyl of R54₂ and the side chain carboxyl of E57₂ are shown as dashed lines, with the distance indicated in angstroms. Residues I29₁, R54₂, V55₂, K58₂, and T59₂, indicated on the protein structure, and residues P293₁, K307₁, Y94₂, E97₂, L98₂, L99₂, and A101₂ (not visible) are involved in hydrophobic interactions with 4c (shown in green; note that the cyclohexane part is not displayed). The predicted position of the N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide moiety of 4c is similar to that of TBHQ (shown in purple). 4c-specific hydrophobic interactions were observed between its imidazo[2,1-b]thiazole part and residues P293₁, K307₁, K58₂, and T59₂.