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 60 publications

(57 citation statements)

References 35 publications

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“…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

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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.

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Section: Discussionsupporting

confidence: 90%

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

et al. 2014

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“…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

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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.

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“…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

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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.

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

Cited by 60 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

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