Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems

Many pathogenic bacteria utilize two-component systems consisting of a hist idine protein kinase (HPK) and a response regulator (RR) for signal transdu ction. During the search for novel inhibitors, several chemical series, inc luding benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, an d salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC(50)s) rang ing from 1.9 to >500 mu M and MICs ranging from 0.5 to >16 mu g/ml for gram -positive bacteria. However, additional observations suggested that mechani sms other than HPK inhibition might contribute to antibacterial activity. I n the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and mac romolecular synthesis. At 4x MIG, 17 of 24 compounds compromised the integr ity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC(50)s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular inc orporation of radiolabeled thymidine and uridine >97% in 5 min and amino ac ids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorpora tion had no measurable MIC (>16 mu g/ml). Fifteen of 24 compounds also caus ed hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cyto plasmic membrane. Bacterial killing by these HPK inhibitors may therefore b e due to multiple mechanisms, independent of HPK inhibition.