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.

A new class of inhibitors of the two-component regulatory systems (TCS) of bacteria was discovered based on the salicylanilide screening hits, closantel (1) and tetrachlorosalicylanilide (9). A systematic SAR study versus a model TCS, KinA/Spo0F, demonstrated the importance of electron-attracting substituents in the salicyloyl ring and hydrophobic groups in the anilide moiety for optimal activity. In addition, derivatives 8 and 16, containing the 2, 3-dihydroxybenzanilide structural motif, were potent inhibitors of the autophosphorylation of the KinA kinase, with IC50s of 2.8 and 6. 3 µM, respectively. Compound 8 also inhibited the TCS mediating vancomycin resistance (VanS/VanR) in a genetically engineered Enterococcus faecalis cell line at concentrations subinhibitory for growth. Closantel (1), tetrachlorosalicylanilide (9), and several related derivatives (2, 7, 10, 11, 20) had antibacterial activity against the drug-resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

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Levofloxacin resistance in Streptococcus pneumoniae is rare, requiring at least two mutations in the quinolone resistance-determining region (QRDR) of topoisomerase IV and DNA gyrase. The prevalence of single QRDR mutations in these genes is unknown. Of 9,438 levofloxacin-susceptible pneumococci from the TRUST 4 surveillance study (1999)(2000), 528 strains (MICs of 0.5 to 2.0 g/ml) were selected for analysis. For comparison, 214 levofloxacin-susceptible strains (MICs of 0.5 to 1 g/ml) isolated between 1992 and 1996 were analyzed. Oligonucleotide probe assay and DNA sequencing were used to detect QRDR mutations leading to changes at Ser79 and Asp83 in ParC, Ser81 in GyrA, and Asp435 in ParE, the most frequently found substitutions among levofloxacin-resistant strains. Among the 1992 to 1996 isolates only one strain (levofloxacin MIC, 1 g/ml) had a mutation (Ser79 to Phe in ParC). No single mutations were found among 270 TRUST 4 strains with levofloxacin MICs of 0.5 g/ml. Among 244 strains for which levofloxacin MICs were 1 g/ml, 15 strains (6.1%) had a parC mutation and 3 strains (1.2%) had a parE mutation. Of 14 strains for which levofloxacin MICs were 2 g/ml, 10 strains (71%) had a parC mutation; no parE mutations were found. No gyrA mutations were detected. It was estimated that 4.5% of the 9,438 levofloxacin-susceptible TRUST 4 isolates (MICs, <0.06 to 2 g/ml) had a single parC or parE QRDR mutation. Although there has been an increase in the prevalence of single-step mutants, the increase may have been overestimated due in part to differences in geographical distribution for the two sets of isolates.

Three patients with bacteremic pneumonia caused by multidrug-resistant Streptococcus pneumoniae were treated unsuccessfully with azithromycin. One S. pneumoniae isolate carried a mef determinant for an efflux pump; a second isolate had an erm determinant. All 3 patients were successfully treated with levofloxacin, an antipneumococcal fluoroquinolone.

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Bacterial two-component regulatory systems (TCS) play a pivotal role in the process of infection. These signal transduction systems enable bacterial pathogens to mount an adaptive response and cope with diverse environmental stresses, including nutrient deprivation, antibiotic onslaught and phagocytosis. Interest in these systems as novel bacterial targets has been rekindled by the recent discovery of several essential systems in important Gram-positive and Gram-negative pathogens. Several series of TCS inhibitors derived from broad screening approaches have been reported in the literature, however, most appear to suffer from poor selectivity, excessive protein binding and/or limited bioavailability. Consequently, pharmaceutical chemists have turned to alternate strategies, such as the design of substrate-based inhibitors, the generation of combinatorial libraries and the isolation of natural products, to identify inhibitors with more desirable properties. Recent structural studies of the histidine protein kinase and response regulator proteins that constitute TCS may provide a foundation for a structure-based design approach to TCS inhibitors.

US levofloxacin-resistant S. pneumoniae isolates were rare and most were unrelated with minimal clonal spread, and were associated with multiple QRDR mutations with extensive cross-resistance noted among fluoroquinolones.