Elucidating the molecular target of symmetric bis-benzimidazoles

Various bis-benzimidazole derivatives have been reported to possess activity against Gram-positive pathogens. However, as yet no mechanism of action has been elucidated to account for the antibacterial activity of this class of compounds. A group of symmetric bis-benzimidazoles (BBZ) designed as anti-cancer agents have previously been shown to possess moderate anti-proliferative activity (Seaton A, Higgins C, Mann J et al. Mechanistic and anti-proliferative studies of two novel, biologically active bis-benzimidazoles. Eur J Cancer 2003; 39: 2548-55). Here it is shown that these and related compounds possess antibacterial activity that is superior to their anti-cancer activity. Minimal inhibitory concentrations (MIC) against Staphylococcus aureus including the methicillin-resistant clinical isolate EMRSA-16 were between 0.03 and 0.5 mug/ml. The compounds were also similarly active against the hyper-permeable Escherichia coli strain MC1061. The cytoxicity of the compounds against a mammalian cell line was up to 60-fold lower than the corresponding antibacterial activities indicating a viable therapeutic window. The BBZ compounds were bactericidal against S. aureus and bacteriostatic against E. coli and were seen to induce gross morphological defects that included cell swelling and filamentation in the respective species. Several approaches were used to investigate the mechanism of action of these compounds as antibacterials. Firstly, the transcriptional and proteomic responses to BBZ treatment were analysed. In S. aureus the transcriptional fingerprint was consistent with responses to DNA damage and topoisomerase inhibition. Using in vitro biochemical assays, it was shown that the BBZ compounds inhibit both S. aureus DNA gyrase supercoiling and topoisomerase IV decatenation activities with IC50 values in the range 5-15 muM. This inhibition was shown to operate through both inhibition of binding to DNA and accumulation of single stranded DNA breaks. In contrast the BBZ compounds did not inhibit DNA gyrase isolated from E. coli. The transcriptional responses in MC1061 were instead consistent with a response to amino acid starvation. Using a multi-copy suppression (MCS) approach, the transcriptional regulator AppY was found to confer resistance to the BBZ compounds when expressed in high copy number. Determination of the AppY regulon, and further MCS experiments indicated that this resistance was linked to an up-regulation of the multi-drug efflux complex MdtEF-TolC.