Novel anti-staphylococcal and anti-biofilm properties of two anti-malarial compounds: MMV665953 {1-(3-chloro-4-fluorophenyl)-3-(3,4-dichlorophenyl)urea} and MMV665807 {5-chloro-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide}

Purpose. The treatment of device-related infections is challenging and current anti-microbial compounds have poor antibiofilm activity. We aimed to identify and characterize novel compounds effective in the eradication of Staphylococcus aureus biofilms. Methodology. Two novel compounds, MMV665953 {1-(3-chloro-4-fluorophenyl)-3-(3,4-dichlorophenyl)urea} and MMV665807 {5-chloro-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide}, effective in killing S. aureus biofilms, were identified by screening of the open access ’malaria box’ chemical library. The minimum bactericidal concentrations, half-maximal inhibition concentration (IC50) values and minimal biofilm killing concentrations effective in the killing of biofilm were determined against meticillin-resistant S. aureus and meticillin-sensitive S. aureus. Fibrin-embedded biofilms were grown under in vivo-relevant conditions, and viability was measured using a resazurin-conversion assay and confocal microscopy. The potential for the development of resistance and cytotoxicity was also assessed. Results. MMV665953 and MMV665807 were bactericidal against S. aureus isolates. The IC₅₀ against S. aureus biofilms was at 0.15-0.58mgl^-1 after 24 h treatment, whereas the concentration required to eradicate all tested biofilms was 4 mgl^-1, making the compounds more bactericidal than conventional antibiotics. The cytotoxicity against human keratinocytes and primary endothelial cells was determined as IC50 7.47 and 0.18mgl^-1 for MMV665953, and as 1.895 and 0.076mgl^-1 for MMV665807. Neither compound was haemolytic nor caused platelet activation. MMV665953 and MMV665807 derivatives with reduced cytotoxicity exhibited a concomitant loss in anti-staphylococcal activity. Conclusion. MMV665953 and MMV665807 are more bactericidal against S. aureus biofilms than currently used antistaphylococcal antibiotics and represent a valuable structural basis for further investigation in the treatment of staphylococcal biofilm-related infections.