Novel piperidinyloxy oxazolidinone antimicrobial agents.

Oxazolidinone antibacterial agents, where the N-substituted piperazinyl group of eperezolid was replaced with a N-substituted piperidinyloxy moiety, were synthesized and shown to be active against a variety of resistant and susceptible Gram-positive organisms. The effect of ring size, positional isomerism, and fluorine substitution on antibacterial activity was examined.

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 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.

Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria.

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).

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

SAR studies of diaryltriazoles against bacterial two-component regulatory systems and their antibacterial activities.

A series of diaryltriazole analogs was discovered to inhibit bacterial two-component regulatory systems in our primary assays, KinA/Spo0F and NRII/NRI. They also showed inhibitory activity in whole cell mechanism-based assays, and they possessed potent activities against several strains of Gram-positive pathogenic bacteria in the standard MIC broth assay.

Comparison of the postantibiotic and postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae.

The postantibiotic subminimum inhibitory concentration effect (PA SME) may simulate in vivo drug exposure more accurately than the postantibiotic effect (PAE) since subinhibitory concentrations of drug persist between antibiotic dosings. In this study, we compared the PAEs and PA SMEs of levofloxacin and ciprofloxacin for clinical isolates of fluoroquinolone-susceptible Staphylococcus aureus and Streptococcus pneumoniae. At two times the MIC, PAEs of levofloxacin were an average of 0.6 h longer than the PAEs obtained for ciprofloxacin for methicillin-susceptible and methicillin-resistant S. aureus strains. The PAEs of levofloxacin and ciprofloxacin ranged from 1.8 to 3.1 and 1.1 to 2.4 h, respectively. Continued exposure of the methicillin-resistant strain to 1/16, 1/8, and 1/4 the MIC resulted in PA SMEs of 6.5, 15.3, and >22.3 h, respectively, for levofloxacin and 3.8, 8.0, and 12.3 h, respectively, for ciprofloxacin. For isolates of S. pneumoniae, at two times the MIC of both fluoroquinolones, the average PAEs of levofloxacin and ciprofloxacin were equivalent: 1.3 h for the penicillin-susceptible isolate and 0.6 h for the penicillin-resistant isolate. Continued exposure of the penicillin-susceptible S. pneumoniae strain to 1/16, 1/8, and 1/4 the MIC resulted in average PA SMEs of 1.0, 1.4, and 2.8 h, respectively, for levofloxacin and 1.8, 2.0, and 2.5 h, respectively, for ciprofloxacin. Continued exposure of penicillin-resistant S. pneumoniae to 1/16, 1/8, and 1/4 the MIC of the same fluoroquinolones resulted in average PA SMEs of 0.6, 1.1, and 2.9 h, respectively, for levofloxacin and 0.6, 1.1, and 1.5 h, respectively, for ciprofloxacin. The PA SMEs observed demonstrate the superior activity of levofloxacin against methicillin-susceptible or methicillin-resistant S. aureus. Although PAEs were similar for the penicillin-susceptible and penicillin-resistant S. pneumoniae strains, the PA SME of levofloxacin at one-fourth the MIC was longer for penicillin-resistant S. pneumoniae.

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.

Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group.

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

Avirulent Salmonella typhimurium delta cya delta crp oral vaccine strains expressing a streptococcal colonization and virulence antigen.

Salmonella typhimurium SR-11 strains lacking adenylate cyclase and the cyclic AMP receptor protein (CRP) due to deletion (delta) mutations in the cya and crp genes, respectively, are avirulent for mice and induce high level protective immunity against subsequent challenge with wild-type virulent S. typhimurium SR-11 cells. The avirulence of these delta cya delta crp mutants has been enhanced by elimination of the 100 kb virulence plasmid pStSR100 without impairing immunogenicity. The present report confirms the avirulence and immunogenicity of these mutant strains, demonstrates that immunization of both four- and eight-week-old mice has no adverse effect on weight gain, and that immunity lasts at least ninety days following initial immunization. Avirulent S. typhimurium strains have been endowed with the ability to produce several streptococcal colonization and virulence antigens for the purpose of constructing recombinant bivalent oral vaccine strains. Important antigenic determinants of the Streptococcus sobrinus surface protein antigen A (SpaA), presumed to be a critical colonization antigen of S. sobrinus, are expressed at high level by the delta cya delta crp S. typhimurium strains. The recombinant vaccine strains are stable in vitro and in animals (for a period of at least eight days) where they localize to the gut-associated lymphoid tissue (GALT).