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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

Mutation analysis verified the binding sites of policresulen against DENV2NS2B/NS3 protease. (A,B,C) IC50 of policresulen against NS2B/N33 (Q106G), NS2B/NS3 (R133G) and NS2B/NS3 (Q106G/R133G) were determined as (A) 4.99 μg/mL; (B) 4.3 μg/mL; (C) 60.8 μg/mL, respectively. (D,E,F) Biacore sensorgrams were obtained from injections of policresulen at 2.94 μg/mL over the immobilized. (D) NS2B/N33 (Q106G). (E) NS2B/NS3 (R133G) or (F) NS2B/NS3 (Q106G/R133G). The sensorgram of policresulen at 2.94 μg/mL binding to wild type NS2B/NS3 was shown in dashed line as a control.
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fig7: Mutation analysis verified the binding sites of policresulen against DENV2NS2B/NS3 protease. (A,B,C) IC50 of policresulen against NS2B/N33 (Q106G), NS2B/NS3 (R133G) and NS2B/NS3 (Q106G/R133G) were determined as (A) 4.99 μg/mL; (B) 4.3 μg/mL; (C) 60.8 μg/mL, respectively. (D,E,F) Biacore sensorgrams were obtained from injections of policresulen at 2.94 μg/mL over the immobilized. (D) NS2B/N33 (Q106G). (E) NS2B/NS3 (R133G) or (F) NS2B/NS3 (Q106G/R133G). The sensorgram of policresulen at 2.94 μg/mL binding to wild type NS2B/NS3 was shown in dashed line as a control.

Mentions: The mutants Q106G, R133G and Q106G/R133G were expressed and purified using an approach similar to that used for the wild-type protein, and enzymatic assays indicated that mutagenesis of either of these two residues exhibited no effects on the enzymatic activity of the protease (Figure S4). As shown in Figure 7A-C, mutation of either of these two residues caused a large decrease in the inhibitory activity of policresulen against the protease, as is indicated by the increases in the IC50 values for the mutants (IC50: Q106G, 4.99 μg/mL; R133G, 4.3 μg/mL; and Q106G/R133G, 60.8 μg/mL). Notably, the double site-directed mutagenesis (Q106G/R133G) rendered the greatest influence on the inhibition of the protease by policresulen, given that it had the largest IC50 value among the three mutants. These results all demonstrate that residues Gln106 and Arg133 are responsible for policresulen inhibition of the DENV2 NS2B/NS3 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)

Mutation analysis verified the binding sites of policresulen against DENV2NS2B/NS3 protease. (A,B,C) IC50 of policresulen against NS2B/N33 (Q106G), NS2B/NS3 (R133G) and NS2B/NS3 (Q106G/R133G) were determined as (A) 4.99 μg/mL; (B) 4.3 μg/mL; (C) 60.8 μg/mL, respectively. (D,E,F) Biacore sensorgrams were obtained from injections of policresulen at 2.94 μg/mL over the immobilized. (D) NS2B/N33 (Q106G). (E) NS2B/NS3 (R133G) or (F) NS2B/NS3 (Q106G/R133G). The sensorgram of policresulen at 2.94 μg/mL binding to wild type NS2B/NS3 was shown in dashed line as a control.
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fig7: Mutation analysis verified the binding sites of policresulen against DENV2NS2B/NS3 protease. (A,B,C) IC50 of policresulen against NS2B/N33 (Q106G), NS2B/NS3 (R133G) and NS2B/NS3 (Q106G/R133G) were determined as (A) 4.99 μg/mL; (B) 4.3 μg/mL; (C) 60.8 μg/mL, respectively. (D,E,F) Biacore sensorgrams were obtained from injections of policresulen at 2.94 μg/mL over the immobilized. (D) NS2B/N33 (Q106G). (E) NS2B/NS3 (R133G) or (F) NS2B/NS3 (Q106G/R133G). The sensorgram of policresulen at 2.94 μg/mL binding to wild type NS2B/NS3 was shown in dashed line as a control.
Mentions: The mutants Q106G, R133G and Q106G/R133G were expressed and purified using an approach similar to that used for the wild-type protein, and enzymatic assays indicated that mutagenesis of either of these two residues exhibited no effects on the enzymatic activity of the protease (Figure S4). As shown in Figure 7A-C, mutation of either of these two residues caused a large decrease in the inhibitory activity of policresulen against the protease, as is indicated by the increases in the IC50 values for the mutants (IC50: Q106G, 4.99 μg/mL; R133G, 4.3 μg/mL; and Q106G/R133G, 60.8 μg/mL). Notably, the double site-directed mutagenesis (Q106G/R133G) rendered the greatest influence on the inhibition of the protease by policresulen, given that it had the largest IC50 value among the three mutants. These results all demonstrate that residues Gln106 and Arg133 are responsible for policresulen inhibition of the DENV2 NS2B/NS3 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