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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 efficiently inhibited the replication of DENV2. (A) BHK-21 cells transfected with Rlu-DENV-Rep were treated with different concentrations of policresulen for 48 h. The cells were harvested, and luciferase activities were measured using Renilia Luciferase Assay Kit. (B) The inhibitory rate of policresulen at different concentrations against DENV2 replicon was converted from luciferase activity.
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fig2: Policresulen efficiently inhibited the replication of DENV2. (A) BHK-21 cells transfected with Rlu-DENV-Rep were treated with different concentrations of policresulen for 48 h. The cells were harvested, and luciferase activities were measured using Renilia Luciferase Assay Kit. (B) The inhibitory rate of policresulen at different concentrations against DENV2 replicon was converted from luciferase activity.

Mentions: Given that policresulen has been determined to inhibit the DENV2 NS2B/NS3 protease, we next evaluated its potential antiviral activity against DENV2 on the cellular level. Rluc-DENV2-Rep which includes a reporter gene (Renilla luciferase, Rluc) and the full genome of DENV2 (pACYC-DENV2-Rluc2A replicon) were used to examine the antiviral effects of policresulen42. In this assay, DENV2 RNA, which included an Rluc gene transcribed from Rluc-DENV2-Rep, was transfected into BHK-21 cells, and the ability of the virus to replicate was monitored by detecting the activity of Renilla luciferase at 48 h post-infection. As indicated in Figure 2A and B, policresulen could effectively inhibit the luciferase activity in a dose-dependent manner, with an IC50 of 4.99 μg/mL. This result, combined with that from the cytotoxicity assay, shows a high therapeutic index (TI) for policresulen of 92.07.


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 efficiently inhibited the replication of DENV2. (A) BHK-21 cells transfected with Rlu-DENV-Rep were treated with different concentrations of policresulen for 48 h. The cells were harvested, and luciferase activities were measured using Renilia Luciferase Assay Kit. (B) The inhibitory rate of policresulen at different concentrations against DENV2 replicon was converted from luciferase activity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561973&req=5

fig2: Policresulen efficiently inhibited the replication of DENV2. (A) BHK-21 cells transfected with Rlu-DENV-Rep were treated with different concentrations of policresulen for 48 h. The cells were harvested, and luciferase activities were measured using Renilia Luciferase Assay Kit. (B) The inhibitory rate of policresulen at different concentrations against DENV2 replicon was converted from luciferase activity.
Mentions: Given that policresulen has been determined to inhibit the DENV2 NS2B/NS3 protease, we next evaluated its potential antiviral activity against DENV2 on the cellular level. Rluc-DENV2-Rep which includes a reporter gene (Renilla luciferase, Rluc) and the full genome of DENV2 (pACYC-DENV2-Rluc2A replicon) were used to examine the antiviral effects of policresulen42. In this assay, DENV2 RNA, which included an Rluc gene transcribed from Rluc-DENV2-Rep, was transfected into BHK-21 cells, and the ability of the virus to replicate was monitored by detecting the activity of Renilla luciferase at 48 h post-infection. As indicated in Figure 2A and B, policresulen could effectively inhibit the luciferase activity in a dose-dependent manner, with an IC50 of 4.99 μg/mL. This result, combined with that from the cytotoxicity assay, shows a high therapeutic index (TI) for policresulen of 92.07.

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