Identification of a novel inhibitor targeting influenza A virus group 2 hemagglutinins

Abstract

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. The putative antiviral therapeutics are useful for the treatment of influenza, however, the emerging resistant strains necessitate a constant search for new drug candidates. Here we report the discovery of a novel antiviral agent, compound CBS1194, which was identified by a parallel high-throughput screening (HTS) campaign using two retroviral pseudotypes bearing H7 or H5 hemagglutinins (HAs). Subsequent analyses demonstrated that CBS1194 is specific to IAVs of group 2, while it has no effect against those of group 1. In a time-of-addition assay, CBS1194 showed a significant inhibitory effect during the early phase of viral infection. In addition, HA-mediated hemolysis can be inhibited by CBS1194 treatment, indicating that this compound may target the HA stalk region, which is responsible for membrane fusion. Escape mutant analyses and in silico docking further revealed that CBS1194 fits into a pocket near the fusion peptide, causing steric hindrance that blocks the low-pH induced rearrangement of HA. In summary, our study identifies a novel fusion inhibitor of group 2 IAVs, which has the potential as lead compound for further development.

Keywords: Fusion inhibitor; Group 2 specific; Hemagglutinin; Influenza A virus.

Fig. 1.. Time-of-addition assay of CBS1194 against influenza A/Hong Kong/1/1968 (H3N2) virus.

MDCK cells grown in 24-well plates were inoculated with influenza A/Hong Kong/1/1968 (H3N2) virus at an moi of 0.01 PFU/cell.After 1 h of virus absorption on ice, culture wells were washed with PBS and fresh medium containing 2 μg/ml of TPCK-trypsin was added, followed by transferring the plate to 37 °C for incubation (Set as 0 h p.i.). CBS1194 or DMSO was added at −1 to 0 (absorption), −1 to 2, 0 to 2, 2 to 4, 4 to 6, 6 to 8, and −1 to 8 h p.i. at a final concentration of 10 μM. Virus yields were determined by measuring TCID₅₀ at 24 h p.i. The data shows the mean and SD of three independent experiments. *, p < 0.05; ***, p < 0.001; student’s t-test.

Fig. 2.. CBS1194 inhibited HA-mediated hemolysis of chicken erythrocytes.

(A) Hemagglutination inhibition assay. CBS1194 or DMSO were mixed with 2-fold serially diluted influenza A/Hong Kong/1/1968 (H3N2) virus and incubated at room temperature for 1 h. Freshly prepared chicken erythrocytes were then added and incubated at room temperature for another 1 h. The final concentration of CBS1194 is 10 μM. (B) Hemolysis inhibition assay. Increasing concentrations of CBS1194 were mixed with influenza A/Hong Kong/1/1968 (H3N2) virus followed by adding a suspension of freshly prepared chicken erythrocytes. The mixture was then acidified to pH 5.0 and incubated at 37 °C for 30 min. After a brief spin, supernatants containing released hemoglobin were transferred to a new plate for measuring OD₅₄₀. As a control, CBS1194 without virus was added to the cells followed by acidification to measure compound induced hemolysis. Data points show the mean and SD of three independent experiments.

Fig. 3.. Trypsin protection assay.

(A) Influenza A/ Hong Kong/1/1968 (H3N2) virus was incubated with either DMSO or different concentrations of CBS1194 followed by acidification. Acidified HA undergoes a change of conformation and exposes an internal trypsin cleavage site within HA. The mixture was then neutralized and 2 μg of trypsin was added. Samples were further processed for western blot using H3 HA specific antibodies. Virus treated with either low pH or trypsin alone served as controls. (B) 20 μM of CBS1194 was added to influenza A/Hong Kong/1/1968 (H3N2) virus before or after acidification, followed by neutralization, trypsin digestion and western blotting. * Cleaved HA1 or HA2.

Fig. 4.. Resistance of escape mutants to CBS1194.

Wildtype or escape mutants of influenza A/Hong Kong/1/1968 (H3N2) virus were used to infect MDCK cells at an moi of 0.01 PFU/cell, in the presence of increasing concentrations of CBS1194. At 24 h postinfection, virus yields were titrated and normalized to the DMSO control. Data points are the mean and SD of two independent experiments. The EC₉₀s were calculated by four-parameter dose-response curve-fitting in GraphPad.

Fig. 5.. In silico docking and predicted binding pocket of CBS1194 within the H3 HA protein structure.

(A) CBS1194 docking site in the HA trimer of A/Hong Kong/1/1968 (H3N2) virus (PDB code: 5THF) was predicted by AutoDock Vina. HA1 T30 and HA2 K117 where CBS1194 escape mutations occurred are indicated by arrows. (B) Surface and (C) Ribbon representations of the predicted binding pocket of CBS1194. The ionized residues surrounding the pocket are shown in yellow, except for K117₂ which is shown in pink. Chains B and D of H3 trimer are shown in cyan and green respectively, while chains A, C, E and F are shown in grey. The fusion peptide within chain D was shown in gold. The brackets indicate the chains of HA. FP, fusion peptide.