Limits...
Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells.

Wu DW, Mao F, Ye Y, Li J, Xu CL, Luo XM, Chen J, Shen X - Acta Pharmacol. Sin. (2015)

Bottom Line: In our in-house library of old drugs (~1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL.Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells.The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China.

ABSTRACT

Aim: Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action.

Methods: An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis.

Results: In our in-house library of old drugs (~1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC50 of 4.99 μg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site-directed mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding.

Conclusion: Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.

No MeSH data available.


Related in: MedlinePlus

Putative binding mode of policresulen interacting with DENV2 NS2B/NS3 protease. (A) Structure of the major active constituent of policresulen. (B) Binding pose of policresulen at the substrate binding site of DENV2 NS2B/NS3 protease. Hydrogen bonds were shown as pink dotted lines. The poses were prepared using PyMol (http://pymol.sourceforge.net/). (C) Ligplots showed the hydrogen-bond and hydrophobic interactions between policresulen and DENV2 NS2B/NS3 protease. (D) The DENV2 NS2B/NS3 protease surface was colored according to electrostatic potential. The binding subareas were labeled as P1, P2 and P3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4561973&req=5

fig6: Putative binding mode of policresulen interacting with DENV2 NS2B/NS3 protease. (A) Structure of the major active constituent of policresulen. (B) Binding pose of policresulen at the substrate binding site of DENV2 NS2B/NS3 protease. Hydrogen bonds were shown as pink dotted lines. The poses were prepared using PyMol (http://pymol.sourceforge.net/). (C) Ligplots showed the hydrogen-bond and hydrophobic interactions between policresulen and DENV2 NS2B/NS3 protease. (D) The DENV2 NS2B/NS3 protease surface was colored according to electrostatic potential. The binding subareas were labeled as P1, P2 and P3.

Mentions: Molecular modeling To better understand the method of policresulen binding to the NS2B/NS3 protease, a 3D binding model of policresulen in the substrate binding site of the protease was investigated by molecular docking. For docking analysis, a protein structural model was prepared using the representative conformer of the NMR structure of the DENV2 NS2B/NS3 protease (PDB ID: 2M9Q), and all amino acid numberings (Figure 5) were based on the sequence of recombinant DENV2 NS2B/NS3 protease. Given that the compound with the structure in Figure 6A is suggested to be one of the major active components of policresulen (PubChem CID: 3050404)48, this structure for policresulen was thus used for docking analysis. As illustrated in Figure 6B–D, on the left side of policresulen, the oxygen of the sulfo group formed a hydrogen bond with the Gln106 side chain in the P1 region of the protease, and the phenol group formed a hydrogen bond with the carbonyl group of the main chain of Gln114. In the middle section of policresulen, the benzene moiety formed hydrophobic interactions with residues Ile109, Ile115, and Val131 in the hydrophobic P2 region of the protease, and the sulfo-group may exhibit electrostatic interactions with His130. On the right side of policresulen, the oxygen of the sulfo-group formed hydrogen bonds with the side chains of residues Thr132 and Arg133 in the positive P3 region of the protease.


Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells.

Wu DW, Mao F, Ye Y, Li J, Xu CL, Luo XM, Chen J, Shen X - Acta Pharmacol. Sin. (2015)

Putative binding mode of policresulen interacting with DENV2 NS2B/NS3 protease. (A) Structure of the major active constituent of policresulen. (B) Binding pose of policresulen at the substrate binding site of DENV2 NS2B/NS3 protease. Hydrogen bonds were shown as pink dotted lines. The poses were prepared using PyMol (http://pymol.sourceforge.net/). (C) Ligplots showed the hydrogen-bond and hydrophobic interactions between policresulen and DENV2 NS2B/NS3 protease. (D) The DENV2 NS2B/NS3 protease surface was colored according to electrostatic potential. The binding subareas were labeled as P1, P2 and P3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4561973&req=5

fig6: Putative binding mode of policresulen interacting with DENV2 NS2B/NS3 protease. (A) Structure of the major active constituent of policresulen. (B) Binding pose of policresulen at the substrate binding site of DENV2 NS2B/NS3 protease. Hydrogen bonds were shown as pink dotted lines. The poses were prepared using PyMol (http://pymol.sourceforge.net/). (C) Ligplots showed the hydrogen-bond and hydrophobic interactions between policresulen and DENV2 NS2B/NS3 protease. (D) The DENV2 NS2B/NS3 protease surface was colored according to electrostatic potential. The binding subareas were labeled as P1, P2 and P3.
Mentions: Molecular modeling To better understand the method of policresulen binding to the NS2B/NS3 protease, a 3D binding model of policresulen in the substrate binding site of the protease was investigated by molecular docking. For docking analysis, a protein structural model was prepared using the representative conformer of the NMR structure of the DENV2 NS2B/NS3 protease (PDB ID: 2M9Q), and all amino acid numberings (Figure 5) were based on the sequence of recombinant DENV2 NS2B/NS3 protease. Given that the compound with the structure in Figure 6A is suggested to be one of the major active components of policresulen (PubChem CID: 3050404)48, this structure for policresulen was thus used for docking analysis. As illustrated in Figure 6B–D, on the left side of policresulen, the oxygen of the sulfo group formed a hydrogen bond with the Gln106 side chain in the P1 region of the protease, and the phenol group formed a hydrogen bond with the carbonyl group of the main chain of Gln114. In the middle section of policresulen, the benzene moiety formed hydrophobic interactions with residues Ile109, Ile115, and Val131 in the hydrophobic P2 region of the protease, and the sulfo-group may exhibit electrostatic interactions with His130. On the right side of policresulen, the oxygen of the sulfo-group formed hydrogen bonds with the side chains of residues Thr132 and Arg133 in the positive P3 region of the protease.

Bottom Line: In our in-house library of old drugs (~1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL.Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells.The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China.

ABSTRACT

Aim: Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action.

Methods: An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis.

Results: In our in-house library of old drugs (~1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC50 of 4.99 μg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site-directed mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding.

Conclusion: Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.

No MeSH data available.


Related in: MedlinePlus