Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells

Rothan, Hussin A.; Abdulrahman, Ammar Y.; Sasikumer, Pottayil G.; Othman, Shatrah; Rahman, Noorsaadah Abd.; Yusof, Rohana

doi:10.1155/2012/251482

Cited by 59 publications

(57 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Besides, the data of this study also confirmed our previous findings of the inhibition potential of PG1, PLSN against dengue virus replication [13], [16]. It has been shown that these peptides exhibited similar antiviral activities against different viruses.…”

Section: Discussionsupporting

confidence: 90%

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

et al. 2014

Self Cite

View full text Add to dashboard Cite

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

show abstract

Section: Discussionsupporting

confidence: 90%

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…To date, several DENV proteases have been identified as potential targets for the discovery of anti-DENV inhibitors, including NS2B/NS3, NS4B and NS5 [49][50][51] , and the NS2B/NS3 protease is particularly important because of its essential role in the replication of DENV [52] . In fact, an inhibition strategy involving the protease complex and facilitated by a co-factor enzyme has been successfully applied in the discovery of antiviral drugs against human immunodeficiency virus (HIV) and hepatitis C virus (HCV) [53] , and several inhibitors targeting the HCV NS3/4A protease have already entered into clinical trials [54][55][56] .…”

Section: Discussionmentioning

confidence: 99%

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

Mao

et al. 2015

Acta Pharmacol Sin

View full text Add to dashboard Cite

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 IC 50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC 50 of 4.99 μg/mL, whereas its IC 50 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 sitedirected 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.

show abstract

“…The activity of recombinant NS2B-NS3pro was measured by a fluorescence-labelled substrate as previously described [23,27]. The function of dengue protease depends on the binding between NS2B and NS3pro to form NS2B-NS3pro complex.…”

Section: Discussionmentioning

confidence: 99%

“…The bioassay used in this study was published by Rothan and co-workers [23]. In brief, endpoint reaction mixtures with total volume of 200 μl were prepared consisted of 20 μM fluorogenic peptide substrate (Boc-Gly-Arg-Arg-AMC), 2 μM of recombinant NS2B-NS3pro, with or without RC-1 of varying concentrations, buffered at pH 8.5 with 200 mM Tris–HCl.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1

et al. 2012

Self Cite

View full text Add to dashboard Cite

BackgroundGlobal resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.MethodsDengue NS2B-NS3pro was produced as a recombinant single chain protein in E. coli and purified by Ni+ affinity chromatography. The RC-1 peptide was produced in E. coli and the tri-disulphide bonds were reformed in a diluted alkaline environment. Protease assay was performed using a fluorogenic peptide substrate and measured by fluorescence spectrometry. Real-time PCR was used for quantification of dengue serotype 2 (DENV-2) viral RNA produced in Vero cells.ResultsThe RC-1 peptide inhibited the activity of recombinant NS2B-NS3pro with different values at 50% inhibitory concentration (IC50) which are temperature dependent (28°C, 46.1 ± 1.7 μM; 37°C, 21.4 ± 1.6 μM; 40°C, 14.1 ± 1.2 μM). The presence of RC-1 significantly reduced viral replication in Vero cells infected with DENV-2 at simultaneous treatment after 48 hrs (70%) and 75 hrs (85%). Furthermore, moderate reduction in viral replication was observed at pre-treatment mode after 48 hrs (40%) and 72 hrs (38%) and post-treatment at 48 hrs (30%) and 72 hrs (45%).ConclusionRecombinant RC-1 inhibits DENV-2 replication in Vero cells by interfering with the activity of its serine protease. Thus, we propose that recombinant RC-1 is a potent, cost-effective dengue virus inhibitor. Therefore, it is suitable to consider RC-1 as a new candidate for drug development against dengue infection.

show abstract

Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells

Cited by 59 publications

References 35 publications

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

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

Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1

Contact Info

Product

Resources

About