Single-molecule FRET-based approach for protein-targeted drug discovery

Abstract

Many therapeutic drugs target various proteins involved in diverse biological processes. Among these proteins, type II topoisomerases are critical targets for anticancer and antibacterial chemotherapies, yet the action mechanisms of many type II topoisomerase-targeting drugs have not been fully elucidated. In this regard, the development of rapid and accurate methods to identify the mode of action of potential drug candidates is crucial to improve the efficiency of drug screening and discovery. Here, using type II topoisomerase as a model system, we present a single-molecule fluorescence resonance energy transfer-based drug screening method capable of delineating when and how the drug candidates participate in the entire reaction steps of the target protein. This unique capability has been demonstrated to be applicable to the identification of representative types of widely prescribed drugs targeting type II topoisomerase: etoposide which stabilizes the enzyme-DNA cleavage complex, and bisdioxopiperazines (ICRF-I93) which lock the N-terminal gate of the enzyme into the closed state. Based on this demonstration experiment, we expect that our proposed method will be extended to broad applications in the screening of potent drugs targeting various proteins.

Keywords: Bisdioxopiperazines; Drug screening; Etoposide; Single-molecule fluorescence resonance energy transfer; Type II topoisomerase.

Fig. 1

Single-molecule FRET observations of the action of the 2 representative prescribed drugs targeting topoisomerase II. (A) DNA design and single-molecule FRET method for screening of topo II-targeting drug candidates. The labeling positions for Cy3 and Cy5 are indicated. (B) Representative intensity and FRET time traces showing enzyme binding, DNA bending, and trapping events (gray arrows) after the addition of 20 nM enzyme, and 500 μM etoposide in the presence of 5 mM Mg²⁺. (C) Relative populations of the trapped cleavage complex as a function of incubation time after the addition (left) and removal (right) of 500 μM etoposide. Buffer-containing enzyme and etoposide was introduced into the detection chamber to trap the cleavage complex. Meanwhile, buffer-lacking enzyme and etoposide was injected into the detection chamber to detrap the cleavage complexes by removing free etoposide. The trapping (10.8 minutes) and detrapping (50.2 minutes) time constants were obtained by fitting the data to a single-exponential function. In the analysis to determine trapping time, the plotted data are the means and standard errors derived from independent experiments. (D) Representative Cy3 (green) and Cy5 (red) fluorescence intensity and FRET (blue) time traces showing DNA bending induced by the closed-clamp conformation in the presence of 1 mM AMPPNP and 100 μM ICRF-193. (E) FRET histograms after the injection of 5 nM enzyme with 1 mM AMPPNP (top) or 1 mM ATP and 100 μM ICRF-193 (bottom). (F) Relative populations of DNA bending in the presence of only 5 nM enzyme (left), with 1 mM AMPPNP (middle) or 1 mM ATP and 100 μM ICRF-193 (right).

Fig. 2

Schematic workflow for the development of a single-molecule FRET–based drug screening method for general protein-targeting drug candidates.