Screening and identification of emodin as an EBV DNase inhibitor to prevent its biological functions
Chung-Chun Wu1, Mei-Shu Chen2, Ting-Ying Lee3
1Translational Cell Therapy Center, Department of Medical Research, China Medical University Hospital, No. 2, Yude Rd., North Dist, Taichung City, 40447, Taiwan. ccwu.jim@gmail.com.
Background:
The Epstein-Barr virus (EBV) is a prevalent oncovirus associated with a variety of human illnesses. BGLF5, an EBV DNase with alkaline nuclease (AN) activity, plays important roles in the viral life cycle and progression of human malignancies and has been suggested as a possible diagnostic marker and target for cancer therapy. Methods used conventionally for the detection of AN activity, radioactivity-based nuclease activity assay and DNA digestion detection by gel electrophoresis, are not suitable for screening AN inhibitors; the former approach is unsafe, and the latter is complicated. In the present study, a fluorescence-based nuclease activity assay was used to screen several natural compounds and identify an EBV DNase inhibitor.
Results:
Fluorescence-based nuclease activity assays, in which the DNA substrate is labelled with PicoGreen dye, are cheaper, safer, and easier to perform. Herein, the results of the fluorescence-based nuclease activity assay were consistent with the results of the two conventional methods. In addition, the PicoGreen-labelling method was applied for the biochemical characterisation of viral nucleases. Using this approach, we explored EBV DNase inhibitors. After several rounds of screening, emodin, an anthraquinone derivative, was found to possess significant anti-EBV DNase activity. We verified the efficacy of emodin using the conventional DNA-cleavage assay. Furthermore, using comet assay and micronucleus formation detection, we confirmed that emodin can inhibit DNase-induced DNA damage and genomic instability. Additionally, emodin treatment inhibited EBV production.
Conclusions:
Using a PicoGreen-mediated nuclease activity assay, we successfully demonstrated that emodin has the potential to inhibit EBV DNase nuclease activity. Emodin also inhibits EBV DNase-related biological functions, suggesting that it is a potential inhibitor of EBV DNase.
Insights
Researchers identified emodin as a potent inhibitor of Epstein-Barr virus (EBV) DNase activity. This natural compound effectively blocks viral DNA damage and production, offering a promising avenue for cancer therapy targeting EBV.
Area of Science:
- Virology
- Biochemistry
- Oncology
Background:
- Epstein-Barr virus (EBV) is an oncovirus linked to various human diseases.
- The EBV-encoded BGLF5 protein possesses alkaline nuclease (AN) activity crucial for viral replication and cancer progression.
- Conventional methods for detecting AN activity are unsuitable for inhibitor screening due to safety and complexity concerns.
Purpose of the Study:
- To develop and validate a fluorescence-based assay for screening EBV DNase inhibitors.
- To identify natural compounds with potential inhibitory activity against EBV DNase.
- To evaluate the therapeutic potential of identified inhibitors against EBV-associated malignancies.
Main Methods:
- A fluorescence-based nuclease activity assay using PicoGreen dye was established and validated against conventional methods.
- High-throughput screening of natural compounds was performed to identify EBV DNase inhibitors.
- Biochemical characterization of viral nucleases and assessment of emodin's effects on DNA damage and viral production were conducted.
Main Results:
- The fluorescence-based assay demonstrated high sensitivity and reliability, consistent with traditional methods.
- Emodin, an anthraquinone derivative, was identified as a potent inhibitor of EBV DNase activity.
- Emodin effectively inhibited DNase-induced DNA damage, genomic instability, and EBV production in vitro.
Conclusions:
- A novel, safe, and efficient fluorescence-based assay facilitates the screening of EBV DNase inhibitors.
- Emodin exhibits significant potential as an inhibitor of EBV DNase and its associated biological functions.
- Emodin represents a promising therapeutic candidate for targeting EBV-driven cancers.
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