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. 1999 Jul;43(7):1693-9.
doi: 10.1128/AAC.43.7.1693.

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

J J Hilliard  1 , R M Goldschmidt, L Licata, E Z Baum, K Bush
Affiliations

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

J J Hilliard et al. Antimicrob Agents Chemother. 1999 Jul.

Abstract

Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 microM and MICs ranging from 0.5 to >16 microg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4x MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 microg/ml). Fifteen of 24 compounds also caused 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 cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

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Figures

FIG. 1
FIG. 1
Structures of various chemical classes of TCS inhibitors. Previous designations for selected compounds are RWJ-49815 for T-1 (5), 9 for S-1, 14 for S-2, 1 for S-3, and 23 for S-4 (26).
FIG. 2
FIG. 2
Effect of selected TCS inhibitors on incorporation of [3H]thymidine into DNA (A), [3H]uridine into RNA (B), and a mixture of 3H-amino acids into protein (C). GRM, gramicidin S; PMB, polymyxin B; TET, tetracycline; RIF, rifampin; LFX, levofloxacin.
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