N-[(Thiophen-3-yl)methyl]benzamides as Fusion Inhibitors of Influenza Virus Targeting H1 and H5 Hemagglutinins

Abstract

Novel antiviral drugs are needed to prepare for infections from influenza A virus (IAV). Here, a series of N-[(thiophen-3-yl)methyl]benzamides, which target the hemagglutinin (HA)-mediated fusion process, is reported. The most active compound, VF-57a, displays a 50% effective concentration (EC₅₀) of ∼0.8 μM and an antiviral selectivity index >130 in Madin-Darby canine kidney (MDCK) cells infected with A/H1N1 virus. VF-57a proved to be a strong inhibitor of A/H1N1 and A/H5N1 pseudovirus entry (EC₅₀ values of 0.3 and 0.8 μM, respectively). Cell-cell fusion assays in HA-expressing cells, surface plasmon resonance-based assessment of HA protein refolding, and resistance studies suggested that VF-57a prevents the conformational change of HA at acidic pH. Molecular modeling highlighted the role of the dimethylthiophene moiety and the amide-based tether in anchoring to the binding cavity of HA. Our findings support the further development of this class of IAV fusion inhibitors against A/H1N1 and A/H5N1 viruses.

1. Structures of Reported Inhibitors of IAV HA-Mediated Fusion, Specifically: Arbidol, , M090, OA-10, 4, TBHQ, Spirothiazolidinones, , CBS1194, 8, JNJ4796, (S)-F0045, CBS1117, ING1466, S20, RO5487624, MBX-2456, DICAM, 16, IY640, RL-007 (9d in the Ref ), CL-385319, 20, and 21

1

Surface representation of the HA protein (HA1: gray; HA2: lavender) and the ligand-binding sites of TBHQ and Arbidol (Site A: orange surface) and JNJ4796, (S)-F0045, and CBS1117 (Site B: green surface).

1. Structures of Anti-IAV Compounds 20 and 21 and Newly Designed Hybrid 22

2. Structures of Benzamides with a Mono- or Di-Substituted Phenyl Ring

3. Structures of Benzamides with Tri- and Tetra-Substituted Phenyl Ring

4. Structures of Benzamides Explored for a Topliss Approach of the Benzene Ring

5. Structures of Benzamides Synthesized for Exploring the Influence of the Thiophene Methyl Groups

6. Structures of Benzamides Featuring different Heterocycles on the Right-Hand Side of the Molecule

7. Structures of Benzamides with Alternative Bridges between the Phenyl and Thiophene Rings

2

Dose–response curves for antiviral (A/H1N1) activity of VF-57a (23) and RL-007. (A) Assay in MDCK cells: inhibition of CPE (open circle symbols) and viability of mock-infected cells (open triangle symbols), both based on the MTS assay. (B) Calu-3 cells infected with strain Virg09 and stained for viral nucleoprotein (NP). Data points are the mean values ± SEM (n = 3); curve fitting was performed using GraphPad Prism 10.2.2 software.

3

Dose–response curves for the inhibition of HA-mediated pseudovirus entry and cell–cell fusion. (A) MDCK cells were transduced with luciferase-expressing A/H1N1 and A/H5N1 pseudoviruses in the presence of the compounds. Three days later, luminescence was measured to assess the compounds’ inhibitory effect on pseudovirus entry. (B) Cell–cell fusion assay in H1 HA-expressing HeLa cells. The scheme shows the assay setup. The compounds were present during the 15-min preincubation and 5-min acidic stage. Luminescence was measured 5 h after inducing cell–cell fusion. Data points are the mean ± SEM of three independent experiments. (C) Microscopic images of HeLa cells transfected with H5 HA and briefly exposed to pH 5.3. The compounds were present during the 15-min preincubation and 5-min acidic stage. In panels A and B, curve fitting was performed using GraphPad Prism 10.2.2 software.

4

VF-57a and RL-007 inhibit HA refolding at pH 5.2. SPR was used to measure the binding of H1 HA to stem-directed antibody C179 and head-directed antibody 7B2-32. H1 HA was preincubated with DMSO or compound and then exposed to neutral or low pH prior to SPR analysis. (A) Sensorgrams of a representative experiment. (B) Average binding response of three biological replicates. Statistical significance was analyzed by a two-sided unpaired t-test. Isotype controls yielded RU values lower than 1 and are therefore not shown. RU, resonance units.

5

Location of the HA mutations that Virg09 virus acquired when passaged under VF-57a or RL-007. The left panel shows the X-ray structure of a Virg09-related H1 HA (PDB ID 3M6S); the suffix refers to the HA1 (gray) or HA2 (lavender) chains. Zoom in the top panel: view of the binding site of TBHQ (PDB ID 3EYM) and Arbidol (PDB ID 5T6N) in group 2 (i.e., H3) HA. Zoom in the bottom panel: binding site of antibody C179 (PDB ID 5C0R) and the group 1-specific inhibitors JNJ4796, (S)-F0045, and CBS1117 (PDB IDs: 6CF7, 6WCR, and 6WMZ, respectively).

6

Binding mode of (S)-F0045 to Site B of H1 HA. (A–C) Top panels: Overlay of (S)-F0045 at the end of the MD simulation (yellow sticks) and its crystallographic pose (gray sticks; PDB ID 6WCR) in each of the three HA (Virg09) protomers (shown in green, lavender, and beige). Bottom panels: Superposition of 10 snapshots taken along the last 100 ns of the trajectory. (D) RMSD plots for each HA protomer’s backbone and for each of the three ligands (shown in green, lavender, and beige).

7

Binding mode of VF-57a to Site B of H1 HA. (A–F) Superposition of 20 snapshots (VF-57a shown as yellow sticks) taken every 5 ns along the last 100 ns of the trajectory in each of the three HA (Virg09) protomers (shown in green, lavender, and beige) for the two distinct alignments of VF-57a (A–C and D–F) along the groove. (G, H) RMSD plots for each HA protomer’s backbone and for each of the three ligands (shown in green, lavender, and beige) in the orientation shown in panels (G) A–C and (H) D–F.

8

Binding mode of Arbidol to Site A of H3 HA. (A–C) Top panels: Overlay of Arbidol at the end of the MD simulation (yellow sticks) and its crystallographic pose (gray sticks) in Site A of H3 HA (PDB ID 5T6N). Bottom panels: Superposition of 10 snapshots taken along the last 100 ns of the trajectory. (D) RMSD plots for each HA protomer’s backbone atoms and for each of the three ligands (shown in green, lavender, and beige).

9

Binding mode of VF-57a to Site A of H1 HA. (A–C) Top panels: Final position of VF-57a (yellow sticks) in Site A of the H1 HA (Virg09)-modified homology model. Bottom panels: Superposition of 10 snapshots taken along the last 100 ns of the trajectory. (D) RMSD plots for each HA protomer’s backbone and for each of the three ligands (shown in green, lavender, and beige). (E) Superposition of the three protomers at the end of the MD simulation, showing the adoption of a common pose for the three VF-57a ligands. The dashed line reflects the hydrogen bond interaction formed between the ligand’s amide NH group and the carbonyl oxygen of V55₂. (F) Detailed view of the final pose of VF-57a with Arbidol (gray sticks; PDB ID 5T6N).

10

Structural basis for the viral resistance profile and group 1 specificity of VF-57a. (A) Representation of the binding mode of VF-57a together with the hydrogen bond formed with V55₂ (shown as sticks) and the resistance mutation S54P₂ (shown as spheres). (B) Comparison of selected sequence regions that define the walls of the binding pocket. (C–E) Representation of the binding pose of VF-57a and the location of residue differences (highlighted as red sticks) between H1 HA (C: Virg09), H5 HA (D: FL22), and H3 HA (E: A/Victoria/361/11).