Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor


Abstract

Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PAN) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T''s active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

Keywords: drug repurposing; lifitegrast; virtual screening.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The structure of PA endonuclease. (A) The structure of RNA-dependent RNA polymerase (influenza A virus H5N1, PDB ID: 6QPF). The PA domain is shown in blue. The PB1 domain is shown in white. The PB2 domain is shown in yellow. (B) The cartoon structure of PA N-terminal (blue) (PDB ID: 6FS6) or PA-I38T N-terminal (white) (PDB ID: 6FS7) endonuclease domain complex with the inhibitor BXM. The BXM is shown in yellow. The Mn2+ is indicated with violet spheres. (C) The PA active site pocket complex with the substrate Amp. Amp is shown in yellow. The Mn2+ is indicated with violet spheres.
Figure 2
Figure 2
Compound inhibition of the endonuclease activity of PAN. The chemical structure of lifitegrast (A) and saquinavir (B). For the inhibition assay, different concentrations of the compounds lifitegrast (C) and saquinavir (D) were incubated with the 1.5 μM PAN and 100 ng ssDNA at 37 °C for 1 h. After the digestion, the products were resolved on agarose gel.
Figure 3
Figure 3
Inhibition of the endonuclease activity of PAN-I38T. The chemical structure of lifitegrast (A) and conivaptan (B). For the inhibition assay, different concentrations of compounds lifitegrast (C) and conivaptan (D) were incubated with the 1.5 μM PAN-I38T and 100 ng M13mp18 at 37 °C for 1 h. After the digestion, the products were resolved on agarose gel.
Figure 4
Figure 4
RMSD (A), Rg (B) and RMSF (C) propensities of PAN and PAN-I38T with ligand during molecular dynamic simulation. The highly flexible residue Asn55–Leu71 in the Loop region is colored red. The residue His41, Asp108, Glu119, and Ile120 associated with the active site is colored blue (D) and is stable.
Figure 5
Figure 5
The interaction of lifitegrast within PAN and PAN-I38T active sites. The representative structure is extracted from the largest number of clusters in the system after molecular dynamics simulation. Electrostatic potential surface of PAN (A) or PAN-I38T (B) structure with lifitegrast in the active site pocket. Structure of PAN (C) or PAN-I38T (D) with lifitegrast. Manganese ions are indicated as gray spheres. Lifitegrast is shown using blue sticks. PAN or PAN-I38T are shown as green cartoons. Hydrogen bonds are shown as yellow dashed lines.
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