Abstract
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.
Highlights
Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern
We propose that the recombinant RC-1 could be considered as a candidate therapeutic peptide against dengue virus infection
We have produced RC-1 as a soluble recombinant peptide fused with ten amino acids linker followed by a 6XHis tag (Figure 1A)
Summary
Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Computational studies indicated that disulphide cyclic peptides have potential to inhibit dengue NS2B-NS3pro [15,16]. Our aim was to investigate the potential inhibition of retrocylin-1 (RC-1), a disulphide cyclic peptide, against dengue NS2B-NS3pro. It has been known that RC-1 (GICRCICGRGICRCICGR) is one of theta-defensins analogues which are circular, cationic, tri-disulphide bonded peptides with beta–sheet structure that can protect human peripheral blood lymphocytes from infection by HIV-1 strains [17]. This peptide is encoded in the human genome by a theta-defensins pseudogene [18]. We propose that the recombinant RC-1 could be considered as a candidate therapeutic peptide against dengue virus infection
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