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

Policresulen reduced the stability of DENV2 NS2B/NS3 protease. (A) UV spectra of NS2B/NS3 protease (20 μmol/L) in presence or absence of policresulen (23.52 μg/mL). Blue curve: absorbance spectrum of NS2B/NS3 protease. Red curve: absorbance spectrum of the protease with policresulen. Green curve: absorbance spectrum of the protease with policresulen (subtracting the effect of policresulen). Black curve: absorbance spectrum of policresulen. (B) The thermal stability of NS2B/NS3 protease combined with or without policresulen was evaluated by DSC thermograms. The temperature of the peak of thermo curve represented the thermal transition midpoints (Tm). Tm value of the protease (black curve) was 51.5 °C, and Tm value of the protease with policresulen (red curve) was 51.17°C.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4561973&req=5

fig4: Policresulen reduced the stability of DENV2 NS2B/NS3 protease. (A) UV spectra of NS2B/NS3 protease (20 μmol/L) in presence or absence of policresulen (23.52 μg/mL). Blue curve: absorbance spectrum of NS2B/NS3 protease. Red curve: absorbance spectrum of the protease with policresulen. Green curve: absorbance spectrum of the protease with policresulen (subtracting the effect of policresulen). Black curve: absorbance spectrum of policresulen. (B) The thermal stability of NS2B/NS3 protease combined with or without policresulen was evaluated by DSC thermograms. The temperature of the peak of thermo curve represented the thermal transition midpoints (Tm). Tm value of the protease (black curve) was 51.5 °C, and Tm value of the protease with policresulen (red curve) was 51.17°C.

Mentions: UV spectral analysis It is well-known that proteins have two strong absorption peaks of ∼ 190-250 nm and ∼ 280 nm44,45. Here, there are two strong absorption peaks at 232 and 278 nm in the UV spectra of the DENV2 NS2B/NS3 protease. As shown in Figure 4A, the incubation of policresulen with the protease caused a red shift of 7 nm for the peak at 232 nm and a decrease in the absorbance intensity, while no changes were observed for the absorption peak at 278 nm. These results implied an interaction of policresulen with the DENV2 NS2B/NS3 protease, which is consistent with the result of the SPR assay. The red shift of the absorption peak at 232 nm demonstrated the changed microenvironment of the peptide bond in the DENV2 NS2B/NS3 protease in the presence of policresulen46,47.


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 reduced the stability of DENV2 NS2B/NS3 protease. (A) UV spectra of NS2B/NS3 protease (20 μmol/L) in presence or absence of policresulen (23.52 μg/mL). Blue curve: absorbance spectrum of NS2B/NS3 protease. Red curve: absorbance spectrum of the protease with policresulen. Green curve: absorbance spectrum of the protease with policresulen (subtracting the effect of policresulen). Black curve: absorbance spectrum of policresulen. (B) The thermal stability of NS2B/NS3 protease combined with or without policresulen was evaluated by DSC thermograms. The temperature of the peak of thermo curve represented the thermal transition midpoints (Tm). Tm value of the protease (black curve) was 51.5 °C, and Tm value of the protease with policresulen (red curve) was 51.17°C.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Policresulen reduced the stability of DENV2 NS2B/NS3 protease. (A) UV spectra of NS2B/NS3 protease (20 μmol/L) in presence or absence of policresulen (23.52 μg/mL). Blue curve: absorbance spectrum of NS2B/NS3 protease. Red curve: absorbance spectrum of the protease with policresulen. Green curve: absorbance spectrum of the protease with policresulen (subtracting the effect of policresulen). Black curve: absorbance spectrum of policresulen. (B) The thermal stability of NS2B/NS3 protease combined with or without policresulen was evaluated by DSC thermograms. The temperature of the peak of thermo curve represented the thermal transition midpoints (Tm). Tm value of the protease (black curve) was 51.5 °C, and Tm value of the protease with policresulen (red curve) was 51.17°C.
Mentions: UV spectral analysis It is well-known that proteins have two strong absorption peaks of ∼ 190-250 nm and ∼ 280 nm44,45. Here, there are two strong absorption peaks at 232 and 278 nm in the UV spectra of the DENV2 NS2B/NS3 protease. As shown in Figure 4A, the incubation of policresulen with the protease caused a red shift of 7 nm for the peak at 232 nm and a decrease in the absorbance intensity, while no changes were observed for the absorption peak at 278 nm. These results implied an interaction of policresulen with the DENV2 NS2B/NS3 protease, which is consistent with the result of the SPR assay. The red shift of the absorption peak at 232 nm demonstrated the changed microenvironment of the peptide bond in the DENV2 NS2B/NS3 protease in the presence of policresulen46,47.

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