Single-Molecule FRET based Screening Methodology for Type II Topoisomerase-Targeting Drugs

Abstract

AbstractType II topoisomerases are an important target of anti-cancer and antibacterial drug development, but exact mechanisms of many type II topoisomerase-targeting drugs are not understood well. A fast and accurate way to understand how drug candidates interact with type II topoisomerases is required for efficient drug screening and drug development. In this work, we demonstrated that the unique capability of single-molecule FRET technique to monitor all the key reaction intermediates of the cleavage reaction of type II topoisomerases can be utilized for the screening of the three representative types of drugs: etoposide which inhibit the ligation step, ICRF 187 and I93 which trap the enzyme in the N-gate clamped conformation, and PCA which hinders the binding step of the enzyme. Detailed kinetic analysis further revealed that etoposide binding occurs after the cleavage reaction, and N-gate clamping of the enzyme by ICRF187/193 requires the enzyme-DNA interaction. We expect single-molecule FRET methodology will be a useful screening tool for type II topoisomerase-targeting drugs.