Molecular Dynamics Simulation reveals the mechanism by which the Influenza Cap-dependent Endonuclease acquires resistance against Baloxavir marboxil

Abstract

Baloxavir marboxil (BXM), an antiviral drug for influenza virus, inhibits RNA replication by binding to RNA replication cap-dependent endonuclease (CEN) of influenza A and B viruses. Although this drug was only approved by the FDA in October 2018, drug resistant viruses have already been detected from clinical trials owing to an I38 mutation of CEN. To investigate the reduction of drug sensitivity by the I38 mutant variants, we performed a molecular dynamics (MD) simulation on the CEN-BXM complex structure to analyze variations in the mode of interaction. Our simulation results suggest that the side chain methyl group of I38 in CEN engages in a CH-pi interaction with the aromatic ring of BXM. This interaction is abolished in various I38 mutant variants. Moreover, MD simulation on various mutation models and binding free energy prediction by MM/GBSA method suggest that the I38 mutation precludes any interaction with the aromatic ring of BXA and thereby reduces BXA sensitivity.

Figure 1

2D summary of interaction analysis results of wild-type and mutant CEN-BXA complexes. The interaction pairs that occur during more than 30% of the simulation time are included. (A) Interaction of BXA with CEN (PDB entry: 6FS6), (B) Interaction between BXA and CEN with I38T mutation (PDB entry: 6FS7), C: Interaction between BXA and CEN with I38F mutation, D: Interaction between BXA and CEN with I38M mutation.

Figure 2

Interaction Fraction summary of CEN-BXA contacts. This graph is normalized by the total simulation time. Interaction-fraction values over 1.0 indicate that the residue has multiple contact routes to interact with the ligand. (A) Interaction fraction of BXA with the wild-type CEN protein (PDBID: 6FS6), (B) Interaction fraction of BXA with CEN having the I38T mutation (PDBID: 6FS7), (C) Interaction fraction of BXA with CEN having the I38F mutation, (D) Interaction fraction of BXA with CEN having the I38M mutation.

Figure 3

BXA binding site of representative structures with highest population in MD simulation. (A) Binding site of BXA depicting interaction between the ligand and Ile38 residue in CEN (PDB entry: 6FS6, Population ratio: 0.163), (B) Binding site of BXA depicting interaction between the ligand and Thr38 residue in CEN (PDB entry: 6FS7, Population ratio: 0.434), (C) Binding site of BXA depicting interaction between the ligand and Phe38 residue in CEN (Population ratio: 0.263), (D) Binding site of BXA depicting interaction between the ligand and Met38 residue in CEN (Population ratio: 0.323).

Figure 4

Pharmacophore feature of BXA for binding to CEN as estimated by MD simulation. Red feature: Hydrogen bond acceptor (lone pair), Blue feature: Hydrogen bond donor, Orange feature: Aromatic ring, (A) The BXA binding site in CEN with important residues represented as sticks, BXA and pharmacophore feature. (B) The pharmacophore feature of BXA is displayed.