A T5 Exonuclease-Based Assay for DNA Topoisomerases and DNA Intercalators

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• DNA topoisomerases are crucial enzymes in all organisms, serving as key targets for antibiotics and anticancer drugs.
• Identifying novel topoisomerase inhibitors is vital, but limited high-throughput screening (HTS) assays hinder progress.

Purpose of the Study:

• To establish a novel, low-cost, fluorescence-based HTS assay for identifying DNA topoisomerase inhibitors.
• To validate the assay's utility and identify new inhibitory compounds.

Main Methods:

• Development of a fluorescence-based HTS assay utilizing T5 exonuclease's specific digestion of supercoiled plasmid DNA.
• Validation of the assay by screening a library of 50 compounds against various topoisomerases.
• Assessment of the assay's capability to identify DNA intercalators, a common source of false positives.

Main Results:

• Successfully established and validated a low-cost, fluorescence-based HTS assay for topoisomerase inhibitors.
• The assay effectively distinguishes topoisomerase activity from DNA intercalation.
• A novel compound exhibiting potent inhibition of both human and bacterial DNA topoisomerase I was discovered.

Conclusions:

• The developed T5 exonuclease-based HTS assay provides an accessible and efficient platform for discovering novel topoisomerase inhibitors.
• This assay facilitates the identification of compounds targeting essential enzymes, with potential applications in developing new therapeutics.
• The discovery of a new DNA topoisomerase I inhibitor highlights the assay's effectiveness.