Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor: History
View Latest Version
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects: Allergy
Contributor:
ye wang

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.

  • lifitegrast
  • virtual screening
  • drug repurposing

1. Introduction

Influenza is an infectious disease that causes 300,000 to 500,000 human deaths globally per year [1][2]. The Influenza virus genome is composed of eight negative RNA segments encoded by multiple viral proteins, including PB2, PB1, PA, HA, NP, NA, M1, and NS1 [3][4]. RNA-dependent RNA polymerase (RdRp) is critical for virus RNA transcription and replication and comprises PA, PB1, and PB2 (Figure 1). The N-terminal of PA domain (PAN) contains an endonuclease active pocket and plays a crucial role in influenza virus polymerase activity. Drugs that abolish PAN endonuclease activity or disturb the assembly of RdRp can effectively inhibit the replication of the influenza virus [5]. The PAN endonuclease domain is highly conserved among different influenza virus subtypes, indicating that PAN is a promising broad-spectrum anti-influenza therapeutic target because of its ability to inhibit virus proliferation during the initial mRNA synthesis stage [6].
Molecules 26 07326 g001 550
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.
The three classes of FDA-approved anti-influenza drugs are: neuraminidase inhibitor (oseltamivir and zanamivir), M2-ion channel inhibitor (adamantanes), and PAN endonuclease inhibitor (Baloxavir acid). Several classes of PAN endonuclease inhibitors researched included: 4-substituted 2,4-dioxobutanoic acids and 3,4-substituted 2,6-diketopiperazines [7], flutamide and derivatives [8][9][10], N-hydroxamic acid-scaffold compounds [11], catechins [12], etc. Baloxavir acid, the inhibitor targeted on the PAN endonuclease, was approved by the FDA as an inhibitor of influenza A and B after proving effective in clinical trials [13].
A mutation in the influenza viral genome led to the influenza’s subsequent drug resistance. The hydrophobic interaction residues between PAN endonuclease and Baloxavir acid were disturbed after the Ile 38 mutated to Thr [14]. The I38T substitution in the PAN endonuclease domain is the primary mutant that leads to influenza’s resistance to Baloxavir acid. This resistance reduces the effectiveness of Baloxavir acid as an anti-influenza drug [15]. Using the I38T mutant as the drug screening target may help to develop and refine the next-generation endonuclease inhibitors.
Drug repurposing research can effectively identify new drug repurposing opportunities, quickly expand the drug market, and reveal new commercially valuable uses for existing drugs [16]. Some examples of drugs that have been successfully repurposed include thalidomide [17], sildenafil [18], bupropion [19], and fluoxetine [20]. These drugs are currently used for applications beyond their initially approved therapeutic indications. The combination of in vitro and in silico methods will increasingly be used in the discovery of novel medicine [21].
This research screened the effectivity of PAN endonuclease inhibitors from the FDA-approved database through in silico methods and in vitro experiments. PAN inhibitors can inhibit the entire PAN endonuclease activity. Since the purpose of this study is to identify drugs that overcome resistance, we screened inhibitors that target both PAN and mutant PAN -I38T. Experimental tests have verified that the drugs lifitegrast and saquinavir have an inhibitory effect on PAN, while the drugs lifitegrast and conivaptan have an inhibitory effect on PAN-I38T. In addition, molecular docking shows the interaction mechanism between lifitegrast and the active site of PAN or PAN-I38T. Research suggests that lifitegrast may be a potential anti-flu drug. Finally, the method employed in this work could be utilized as a fast and viable strategy for accelerating research in the treatment of influenza.

2. Analysis on Results

2.1. Virtual Screening and Compounds Selection

To develop the new PAN endonuclease inhibitor structure, we performed the virtual screening protocol based on the PAN and mutant PAN-I38T structure. The residue Ile38 was not essential in either metal-ion binding or in catalytic activity. The I38 substitution does not block the endonuclease reaction but offers resistance to the inhibitor because the I38 side chain interacts with the Baloxavir acid. After the virtual screening, the compounds interacting with PAN or PAN-I38T were sorted by affinity score. The top-ranked compounds listed in Tables S1 and S2 were selected. Nine compounds closely interacted with PAN and PAN-I38T as hit compounds. Finally, six compounds, saquinavir, conivaptan, lifitegrast, rifaximin, dutasteride, and lurasidone, were used for further basic gel endonuclease determination. In comparison, three compounds, Teniposide [22], Simeprevir [23], and Nilotinib [24], were filtered according to levels of cytotoxicity.

2.2. The Inhibitory Effect of PAN or PAN-I38T with Compounds

We identified the five compounds that could inhibit the endonuclease activity of PAN or PAN-I38T, respectively. Next, we incubated different concentrations of compounds with PAN or PAN-I38T and then detected the cleavage of a substrate (Figure 2 and Figure 3).
Molecules 26 07326 g002 550
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.
Molecules 26 07326 g003 550
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.
The results showed that the compound saquinavir could effectively inhibit the cleavage of PAN substrate (Figure 2D), but not PAN-I38T substrate. Meanwhile, compound conivaptan can effectively inhibit the cleavage of PAN-I38T substrate (Figure 3D), but not PAN substrate. However, compounds conivaptan, dutasteride, rifaximin, and lurasidone showed no inhibition of the endonuclease activity of PAN (Figure S1); compounds dutasteride, rifaximin, saquinavir, and lurasidone showed no inhibition of the endonuclease activity of PAN-I38T (Figure S2). Finally, the compound lifitegrast was shown to effectively inhibit the substrate cleavage of both PAN and PAN-I38T at 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively (Table 1).
Table 1. IC50 values of compound inhibited PAN and PAN-I38T endonuclease.
Chemical Name PAN IC50 (μg/mL) 1 PAN-I38T IC50 (μg/mL)
lifitegrast 32.82 ± 1.34 26.81 ± 1.2
conivaptan NI 227.7 ± 1.33
saquinavir 372.7 ± 1.38 NI
1 Measurement IC50 values of compounds through five independent experiments and data were shown as mean ± SD.

 

2.3. Retained Stability of Conformations of PAN/PAN-I38T and Lifitegrast during MD Simulations

To gain further insight into the binding mode of the system after reaching equilibrium, 100 ns MD simulations were performed using an explicit solvent. The initial confirmation of lifitegrast was obtained from the optimal pose of molecular docking operations in virtual screening. The RMSD values are an essential parameter in assessing the stability of a protein–ligand complex. As shown in Figure 4A, the structure of both proteins and ligands acquired good equilibrium during 100.0 ns. Accordingly, we can draw the same conclusion from the radius of the gyration of protein analysis (Figure 4B).
Molecules 26 07326 g004 550
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.
Furthermore, to estimate the structural flexibility, the mean RMSF values were calculated. Figure 4C indicates that residues that interact with lifitegrast at the active pocket of protein remain stable. Although the residue Asn55-Leu71 in the Loop region is highly flexible, the overall protein conformation is stable (Figure 4D).

2.4. The Interaction Mechanism between PAN/PAN-I38T and Lifitegrast

Next, the protein–ligand complex structures in the 100 ns molecular simulation trajectories with similar conformations were divided into the same clusters. The representative frame of the largest cluster was extracted for analysis. Figure 5A,B show that lifitegrast binds to PAN/PAN-I38T at the structure active site and forms hydrogen bond interactions with lifitegrast (Figure 5C,D). Table 2 gives the detailed non-bond parameters of lifitegrast and PAN/PAN-I38T. Comparing the number of hydrogen bonds between protein and lifitegrast, it can be concluded that lifitegrast has three hydrogen-bond interactions with residues Arg124 of PAN, and four hydrogen-bond interactions with residues Trp88, Thr123, and Arg125 of PAN-I38T.
Molecules 26 07326 g005 550
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.
Table 2. Hydrogen bond parameters of lifitegrast and PAN/PAN-I38T.
PAN PAN-I38T
Donors Atom Receptor Atom Distances (Å) 1 Donors Atom Receptor Atom Distances (Å)
Arg124:NH lifitegrast:O4 2.21 Trp88:HE1 lifitegrast:O24 2.77
Arg124:HE lifitegrast:O3 2.02 Thr123:HG1 lifitegrast:O3 2.46
Arg124:1HH2 lifitegrast:O3 2.48 Arg125:HE lifitegrast:O3 2.16
      Arg125:1HH2 lifitegrast:O3 2.56
1 The length of the hydrogen bonds.

This entry is adapted from the peer-reviewed paper 10.3390/molecules26237326

References

  1. Thompson, W.W.; Shay, D.K.; Weintraub, E.; Brammer, L.; Cox, N.; Anderson, L.J.; Fukuda, K. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003, 289, 179–186.
  2. Iuliano, A.D.; Roguski, K.M.; Chang, H.H.; Muscatello, D.J.; Palekar, R.; Tempia, S.; Cohen, C.; Gran, J.M.; Schanzer, D.; Cowling, B.J.; et al. Estimates of global seasonal influenza-associated respiratory mortality: A modelling study. Lancet 2018, 391, 1285–1300.
  3. Rambaut, A.; Pybus, O.G.; Nelson, M.I.; Viboud, C.; Taubenberger, J.K.; Holmes, E.C. The genomic and epidemiological dynamics of human influenza A virus. Nature 2008, 453, 615–619.
  4. De Vlugt, C.; Sikora, D.; Pelchat, M. Insight into Influenza: A Virus Cap-Snatching. Viruses 2018, 10, 641.
  5. Zhou, Z.; Liu, T.; Zhang, J.; Zhan, P.; Liu, X. Influenza A virus polymerase: An attractive target for next-generation anti-influenza therapeutics. Drug Discov. Today 2018, 23, 503–518.
  6. Jones, J.C.; Marathe, B.M.; Lerner, C.; Kreis, L.; Gasser, R.; Pascua, P.N.; Najera, I.; Govorkova, E.A. A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro. Antimicrob. Agents Chemother. 2016, 60, 5504–5514.
  7. Tomassini, J.; Selnick, H.; Davies, M.E.; Armstrong, M.E.; Baldwin, J.; Bourgeois, M.; Hastings, J.; Hazuda, D.; Lewis, J.; McClements, W.; et al. Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds. Antimicrob. Agents Chemother. 1994, 38, 2827–2837.
  8. Tomassini, J.E.; Davies, M.E.; Hastings, J.C.; Lingham, R.; Mojena, M.; Raghoobar, S.L.; Singh, S.B.; Tkacz, J.S.; Goetz, M.A. A novel antiviral agent which inhibits the endonuclease of influenza viruses. Antimicrob. Agents Chemother. 1996, 40, 1189–1193.
  9. Singh, S.B.; Tomassini, J.E. Synthesis of natural flutimide and analogous fully substituted pyrazine-2,6-diones, endonuclease inhibitors of influenza virus. J. Org. Chem. 2001, 66, 5504–5516.
  10. Mikhail, S.B.; Irina, T.F.; Mikhail Yu, S.; Ilia, V.Y. Ring-expanding rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-ones in synthesis of flutimide analogs. Tetrahedron 2014, 70, 3714–3719.
  11. Cianci, C.; Chung, T.D.Y.; Meanwell, N.; Putz, H.; Hagen, M.; Colonno, R.J.; Krystal, M. Identification of N-Hydroxamic Acid and N-Hydroxyimide Compounds that Inhibit the Influenza Virus Polymerase. Antivir. Chem. Chemother. 1996, 7, 353–360.
  12. Kuzuhara, T.; Iwai, Y.; Takahashi, H.; Hatakeyama, D.; Echigo, N. Green tea catechins inhibit the endonuclease activity of influenza A virus RNA polymerase. PLoS Curr. 2009, 1, Rrn1052.
  13. Heo, Y.A. Baloxavir: First Global Approval. Drugs 2018, 78, 693–697.
  14. Jones, J.C.; Kumar, G.; Barman, S.; Najera, I.; White, S.W.; Webby, R.J.; Govorkova, E.A. Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors. MBio 2018, 9, e00430-18.
  15. Checkmahomed, L.; M’Hamdi, Z.; Carbonneau, J.; Venable, M.C.; Baz, M.; Abed, Y.; Boivin, G. Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains. J. Infect. Dis. 2020, 221, 63–70.
  16. Ashburn, T.T.; Thor, K.B. Drug repositioning: Identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 2004, 3, 673–683.
  17. Franks, M.E.; Macpherson, G.R.; Figg, W.D. Thalidomide. Lancet 2004, 363, 1802–1811.
  18. Goldstein, I.; Lue, T.F.; Padma-Nathan, H.; Rosen, R.C.; Steers, W.D.; Wicker, P.A. Oral sildenafil in the treatment of erectile dysfunction. Sildenafil Study Group. N. Engl. J. Med. 1998, 338, 1397–1404.
  19. Jorenby, D.E.; Leischow, S.J.; Nides, M.A.; Rennard, S.I.; Johnston, J.A.; Hughes, A.R.; Smith, S.S.; Muramoto, M.L.; Daughton, D.M.; Doan, K.; et al. A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation. N. Engl. J. Med. 1999, 340, 685–691.
  20. Steiner, M.; Steinberg, S.; Stewart, D.; Carter, D.; Berger, C.; Reid, R.; Grover, D.; Streiner, D. Fluoxetine in the treatment of premenstrual dysphoria. Canadian Fluoxetine/Premenstrual Dysphoria Collaborative Study Group. N. Engl. J. Med. 1995, 332, 1529–1534.
  21. Zhang, J.; Ren, L.; Wang, Y.; Fang, X. In silico study on identification of novel MALT1 allosteric inhibitors. RSC Adv. 2019, 9, 39338–39347.
  22. He, S.; Yang, H.; Zhang, R.; Li, Y.; Duan, L. Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions. Int. J. Pharm. 2015, 478, 131–137.
  23. Li, H.; Tan, J.L.; Li, J.R.; Liu, N.N.; Chen, J.H.; Lv, X.Q.; Zou, L.L.; Dong, B.; Peng, Z.G.; Jiang, J.D. A proof-of-concept study in HCV-infected Huh7.5 cells for shortening the duration of DAA-based triple treatment regimens. Biomed. Pharm. 2019, 116, 108976.
  24. Chen, S.; Liu, G.; Chen, J.; Hu, A.; Zhang, L.; Sun, W.; Tang, W.; Liu, C.; Zhang, H.; Ke, C.; et al. Ponatinib Protects Mice From Lethal Influenza Infection by Suppressing Cytokine Storm. Front. Immunol. 2019, 10, 1393.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
This entry is offline, you can click here to edit this entry!
Video Production Service
Feedback