Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI

Abstract

DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.

Figure 1. Screening the Microsource GenPlus chemical library against E. coli DNA gyrase and M. mazei DNA topoisomerase VI.

A. Results from the DNA gyrase screen. B. Results from the topoisomerase VI screen. Each bar represents the average percentage inhibition for a compound screened in duplicate. Compound bars have been coloured to indicate 96-well plate groupings. The arbitrary hit threshold of 25% inhibition is demonstrated by the dotted lines.

Figure 2. Structures of screen hits.

The M. mazei topo VI inhibitors are circled in blue on the left and the DNA gyrase inhibitors are circled in purple on the right. The 11 already well characterised inhibitors of DNA gyrase identified by the screen are not shown.

Figure 3. In vitro characterisation of DNA gyrase screen hits.

A. Determination of the IC₅₀ for mitoxantrone in a supercoiling assay with 1 unit of gyrase (12 nM); 100 μM ciprofloxacin (Cip.) was used as a positive control for inhibition. The positions of relaxed (Rel.) and negatively supercoiled (SC) DNA are indicated. B. Determination of the IC₅₀ for suramin. C. Assaying the abilities of mitoxantrone and suramin to induce gyrase-mediated DNA cleavage. The reactions were carried out in the absence of ATP. Ciprofloxacin was used as a positive control for gyrase-mediated cleavage. The position of linear DNA (Lin.) is indicated. D. Suramin-induced protection of DNA from Ca²⁺-induced, gyrase-mediated cleavage. E. Inhibition of gyrase binding to a 147 bp DNA fragment by suramin.

Figure 4. Growth of M. smegmatis mc²155 in the presence of mitoxantrone or suramin.

A. Bacteria were grown in liquid cultures in the presence of drug for 22 hours, and then plated onto drug-free agar. After a further 48 h the colonies were counted for each concentration of drug. B. Colony counts for bacteria grown in the presence of 0, 13 or 65 µM mitoxantrone for 16, 19 or 22 hours before being plated onto solid media.

Figure 5. In vitro characterisation of topoisomerase VI screen hits.

A. Determination of IC₅₀ values for hits in the presence of 1 unit (50 nM) of M. mazei topo VI. This resulted in the following IC₅₀ values: 6 μM for 9-aminoacridne; 30 μM for m-amsacrine; 30 μM for suramin; 30 μM for hexylresorcinol; 40 μM for purpurin; 8 μM for quinacrine; and 2 μM for mitoxanthrone. B. Native gel shift assays for the binding of M. mazei topo VI to a 147 bp DNA fragment in the presence of screen hits.

Figure 6. DNA cleavage assays with topoisomerase VI screen hits.

A. Assaying the abilities of screen hits to induce M. mazei topo VI-mediated DNA cleavage with 1 unit topo VI (50 nM). B. Inhibition of S. shibatae topo VI by screen hits. C. Assaying the abilities of screen hits to induce S. shibatae topo VI-mediated DNA cleavage. D. Protection of DNA from ADPNP-induced, S. shibatae topo VI-mediated cleavage by screen hits.

Figure 7. Inhibition of Arabidopsis hypocotyl extension by hexylresorcinol.

All plants were grown for 5 days in the dark. A. Average length of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol. Error bars represent the standard deviation of the samples. B. Percentage germination of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol.

Figure 8. Activity of hexylresorcinol on Arabidopsis thaliana col-0.

A. Hypocotyl extension assay (1 mm scale bars). Left: control plant grown in the absence of hexylresorcinol. Centre: plant grown on 40 μM hexylresorcinol exhibiting normal morphology. Right: plant grown on 40 μM hexylresorcinol exhibiting dwarf morphology. B. Cyro-electron microscopy images of hypocotyls (100 μm white scale bar). Top: control plant grown in absence of hexylresorcinol. Middle: plant grown on 40 μM hexylresorcinol exhibiting normal morphology. Bottom: plant grown on 40 μM hexylresorcinol exhibiting dwarf morphology. C. Three-week-old Arabidopsis plants grown on 40 μM hexylresorcinol. A control plate which did not contain the compound was also included. Photos at a higher magnification were taken of plants displaying different morphologies. Plants 2, 5 and 7 displayed the “dwarf” morphology, whilst plants 1 and 6 were similar to the control in appearance. Plants 3 and 4 appeared intermediate in morphology. All hexylresorcinol-grown plants appeared somewhat transparent compared to the control plants.