An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

<div><p>The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of “induced fit” whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This “induced fit” pocket may be a target for structure-based design of more potent influenza fusion inhibitors.</p></div

Charge Density (<b>ρ</b>_b) and Its Laplacian (<b>∇</b>²<b>ρ</b>_b) at BCPs between Substrate and Main Residues at B3LYP/6-31+G (d,p) Level of Theory (a.u.).

<p>Charge Density (<b>ρ</b>_b) and Its Laplacian (<b>∇</b>²<b>ρ</b>_b) at BCPs between Substrate and Main Residues at B3LYP/6-31+G (d,p) Level of Theory (a.u.).</p

Dynamic stability from molecular docking simulation.

<p>A) The root-mean square deviation (RMSD) values with respect to the initial structures. B) The total energy (ETOT), potential energy (EPTOT),temperature (TEMP) and kinetic energy (EKTOT) fluctuations of the complex with the ligand binding versus simulation time.</p

The initial structure of CL-385319-hemagglutinin complex.

<p>The initial structure of CL-385319-hemagglutinin complex.</p

Hydrogen bonds formed between CL-385319 and residues in binding pocket.

<p>Hydrogen bonds formed between CL-385319 and residues in binding pocket.</p

The expression of HA on the surface of wild-type and mutant pseudoviruses, as determined by Western blotting.

<p>The expression of HA on the surface of wild-type and mutant pseudoviruses, as determined by Western blotting.</p

Site-directed mutagenesis analysis.

<p>A) The infectivities of mutant H5N1 pseudoviruses in MDCK cells. Wild-type pseudovirus was used as the positive control, while Env^- pseudovirus and cells-only (mock) were used as the negative control. B) Inhibitory activity of CL-385319 against the infection of the mutant H5N1 pseudoviruses. The samples were tested in triplicate, and the data were presented in mean ± SD. This experiment was repeated twice with similar results.</p

The lowest energy conformation of CL-385319-hemagglutinin complex.

<p>The lowest energy conformation of CL-385319-hemagglutinin complex.</p

The averaged distance between residue F110₂ and residue M24₁.

<p>A) The initial conformation of CL-385319-hemagglutinin complex. B) The averaged conformation of CL-385319-hemagglutinin complex over MD simulation.</p

Results of the CAS for HA main residues in CL-385319 binding (kcal/mol).

<p>Results of the CAS for HA main residues in CL-385319 binding (kcal/mol).</p