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.