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Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.

ABSTRACT: BACKGROUND: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. 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. METHODS:In 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. RESULTS:We 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. CONCLUSIONS:Our study shows the practicality of a customized biochemical assay to find possible inhibitors of dengue viral protease during the initial stages of drug discovery.

SUBMITTER: Ulanday GE

PROVIDER: S-EPMC4979183 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.

Ulanday Gianne Eduard L GE Okamoto Kenta K Morita Kouichi K

Tropical medicine and health 20160810

<h4>Background</h4>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. 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 ...[more]

PMID: 27551237

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Project description:BackgroundHepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host.AimTo develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.MethodsFull-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.ResultsNS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC50) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98.ConclusionFused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32 µmol/L.

Dataset Information

Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.

Publications

Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.

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