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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

Policresulen as a competitive inhibitor exhibited binding affinity to DENV2 NS2B/NS3 protease. (A) Kinetic analysis of policresulen against NS2B/NS3 protease via double reciprocal plots of 1/V versus 1/[S]. (B) Secondary plot of [S]/V versus different concentrations of policresulen. The inhibitory constant value was then determined using linear regression analysis. (C) Binding ability of policresulen to DENV2 NS2B/NS3 protease. The KD value of policresulen to NS2B/NS3 protease was fitted with 1:1 Langmuir binding model by BiacoreT200 evaluation software. Rmax for policresulen binding to NS2B/NS3 protease was 257.8 Ru.
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fig3: Policresulen as a competitive inhibitor exhibited binding affinity to DENV2 NS2B/NS3 protease. (A) Kinetic analysis of policresulen against NS2B/NS3 protease via double reciprocal plots of 1/V versus 1/[S]. (B) Secondary plot of [S]/V versus different concentrations of policresulen. The inhibitory constant value was then determined using linear regression analysis. (C) Binding ability of policresulen to DENV2 NS2B/NS3 protease. The KD value of policresulen to NS2B/NS3 protease was fitted with 1:1 Langmuir binding model by BiacoreT200 evaluation software. Rmax for policresulen binding to NS2B/NS3 protease was 257.8 Ru.

Mentions: Inhibitor type assay Because policresulen has been determined to be a DENV2 NS2B/NS3 protease inhibitor able to efficiently suppress the replication of DENV2, we next investigated the manner in which it inhibits the DENV2 NS2B/NS3 protease. As shown in Figure 3A, the Lineweaver-Burk plot analysis from the enzymatic inhibition data indicates that policresulen acted as a competitive inhibitor against the DENV2 NS2B/NS3 protease with an inhibitory constant (Ki) value of 0.27 μg/mL (Figure 3B).


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)

Policresulen as a competitive inhibitor exhibited binding affinity to DENV2 NS2B/NS3 protease. (A) Kinetic analysis of policresulen against NS2B/NS3 protease via double reciprocal plots of 1/V versus 1/[S]. (B) Secondary plot of [S]/V versus different concentrations of policresulen. The inhibitory constant value was then determined using linear regression analysis. (C) Binding ability of policresulen to DENV2 NS2B/NS3 protease. The KD value of policresulen to NS2B/NS3 protease was fitted with 1:1 Langmuir binding model by BiacoreT200 evaluation software. Rmax for policresulen binding to NS2B/NS3 protease was 257.8 Ru.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561973&req=5

fig3: Policresulen as a competitive inhibitor exhibited binding affinity to DENV2 NS2B/NS3 protease. (A) Kinetic analysis of policresulen against NS2B/NS3 protease via double reciprocal plots of 1/V versus 1/[S]. (B) Secondary plot of [S]/V versus different concentrations of policresulen. The inhibitory constant value was then determined using linear regression analysis. (C) Binding ability of policresulen to DENV2 NS2B/NS3 protease. The KD value of policresulen to NS2B/NS3 protease was fitted with 1:1 Langmuir binding model by BiacoreT200 evaluation software. Rmax for policresulen binding to NS2B/NS3 protease was 257.8 Ru.
Mentions: Inhibitor type assay Because policresulen has been determined to be a DENV2 NS2B/NS3 protease inhibitor able to efficiently suppress the replication of DENV2, we next investigated the manner in which it inhibits the DENV2 NS2B/NS3 protease. As shown in Figure 3A, the Lineweaver-Burk plot analysis from the enzymatic inhibition data indicates that policresulen acted as a competitive inhibitor against the DENV2 NS2B/NS3 protease with an inhibitory constant (Ki) value of 0.27 μg/mL (Figure 3B).

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