Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems

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 μM and MICs ranging from 0.5 to >16 μg/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 4× 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 μg/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.

Synergistic interactions of nisin in combination with cinnamaldehyde against Staphylococcus aureus in pasteurized milk

“…Therefore, live cells appear green and dead ones appear red. The method of measuring bacterial membrane damage was according to previous study (Hilliard, Goldschmidt, Licata, Baum, & Bush, 1999;Hemaiswarya & Doble, 2010). The S. aureus ATCC 29213 was grown and adjusted to an OD 600 of 0.3, the cell pellet was resuspended to 1/10 of the original volume and then diluted 1:20 into either water or water containing test compounds at 4 Â MIC.…”

Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel

“…Exponentially growing cultures (50 ml) of E. coli (strain BWDK, M9AA-M medium) or S. aureus (strain Newman, CDM-M medium; Hilliard et al, 1999) were incubated with 100-fold MIC of ERY (100 mg/ml for E. coli; 25 mg/ml for S. aureus), TEL (50 mg/ml for E. coli; 12.5 mg/ml for S. aureus), azithromycin (100 mg/ml for E. coli), or solithromycin (50 mg/ml for E. coli) for 15 min. [ 35 S]-methionine (250 mCi, specific activity 1,175 Ci/mmol) was then added, and the cells were incubated for 3 min, followed by the addition of unlabeled L-methionine to final concentration of 80 mg/ml and further incubation for 7 min.…”

Synthesis and antibacterial activity of 5-ylidenethiazolidin-4-ones and 5-benzylidene-4,6-pyrimidinediones

“…In the course of our studies towards the discovery of novel Mur ligase inhibitors, recently described 2H-1,4-benzoxazin-3(4H)-onebased inhibitors of bacterial histidine protein kinase [18,19] attracted our attention due to similarities in the structure of Mur enzymes and various kinases. Thus, compounds 10 and 11, in which the rhodanine moiety is connected to the 2H-1,4-benzoxazin-3(4H)-one scaffold via an ethylidene linker, were prepared as rhodanine analogues of histidine protein kinase inhibitors II and III (Fig.…”