Abstract
BackgroundDengue disease is one of the most significant vector-borne illnesses in the world. The emergence and re-emergence of dengue infections in many parts of the world affect millions annually and continue to burden public health systems especially in low-income populations. Advances in dengue vaccine development showed promising results; however, protection seems to be suboptimal. There is no licensed chemotherapeutic agent against dengue to date. An ideal scenario of combinatorial vaccination of high-risk individuals and chemotherapy of the diseased during outbreaks may compensate for the meager protection offered by the vaccine. The dengue virus protease is important to viral replication and, as such, has been identified as a potential target for antivirals. It is, therefore, our objective to establish and optimize an appropriate screening method for use during the early stages of drug development for dengue.MethodsIn this study, we developed and optimized a biochemical assay system for use in screening compound libraries against dengue virus protease. We tested the selected protease inhibitors with a cell-based assay to determine inhibition of viral replication.ResultsWe have presented direct plots of substrate kinetics data showing an apparent inhibition of the protease at excessive substrate concentrations. The most common sources of interference that may have affected the said observation were elucidated. Finally, a screen was done on an existing compound library using the developed method. The compounds selected in this study showed inhibitory activity against both the recombinant dengue protease and cell-based infectivity assays.ConclusionsOur study shows the practicality of a customized biochemical assay to find possible inhibitors of dengue viral protease during the initial stages of drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s41182-016-0025-6) contains supplementary material, which is available to authorized users.
Highlights
Dengue disease is one of the most significant vector-borne illnesses in the world
Expression, and purification of recombinant DENV2 NS2B3pro The DENV2 NS2B(H)-G4SG4-NS3pro plasmid construct included the hydrophilic residues 49-96 of NS2B and N-terminal 180 amino acids of NS3 joined by a glycine linker (Fig. 1a) [26]
Since the HPLC result was comparable to the purity produced by the crude open column immobilized metal affinity chromatography (IMAC), the latter method was utilized for the remainder of the study
Summary
Dengue disease is one of the most significant vector-borne illnesses in the world. The emergence and re-emergence of dengue infections in many parts of the world affect millions annually and continue to burden public health systems especially in low-income populations. The causative agent, dengue virus, is a single-stranded RNA virus belonging to the Flavivirus genus of the Ulanday et al Tropical Medicine and Health (2016) 44:22 family Flaviviridae It has four serotypes wherein each DENV serotype is phylogenetically distinct, suggesting that each could be considered as a separate virus. Dengue infection is generally asymptomatic, it may result in a wide spectrum of clinical disease, ranging from a mild flu-like syndrome (dengue fever) to the most severe forms of the disease, which are characterized by coagulopathy, increased vascular fragility, and permeability (dengue hemorrhagic fever). The latter may progress to hypovolemic shock (dengue shock syndrome). Age, and timing of infection are some of the aspects to be considered [9]
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