ABSTRACT
d-boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d-Ala-d-Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d-boroAla was identified and characterized as an antibacterial agent. d-boroAla has activity against both Gram-positive and Gram-negative organisms, with minimal inhibitory concentrations down to 8 µgâ/âmL. A structure-function study on the alkyl side chain (NH(2) -CHR-B(OR')(2) ) revealed that d-boroAla is the most effective agent in a series including boroGly, d-boroHomoAla, and d-boroVal. l-boroAla was much less active, and N-acetylation completely abolished activity. An LC-MSâ/âMS assay was used to demonstrate that d-boroAla exerts its antibacterial activity by inhibition of d-Ala-d-Ala ligase. d-boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d-Ala-d-Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d-Ala-d-Ala ligase. d-boroAla demonstrated a frequency of resistance of 8 × 10(-8) at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d-boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d-Ala-d-Ala ligase targeted antibacterial agents. This study also demonstrates d-boroAla as a possible probe for d-Ala-d-Ala ligase function.
Subject(s)
Alanine/analogs & derivatives , Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , Peptide Synthases/antagonists & inhibitors , Alanine/chemistry , Alanine/pharmacology , Anti-Bacterial Agents/chemistry , Bacillus subtilis/drug effects , Boronic Acids/chemistry , Escherichia coli/drug effects , Microbial Sensitivity Tests , Peptide Synthases/metabolism , Salmonella typhimurium/drug effects , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
Bacterial cell wall biosynthesis is the target of several antibacterial agents and is also of interest as a target for future antibacterial agent development. Given the now widespread availability of liquid chromatography-tandem mass spectrometry (LC-MS/MS) instruments, the development of LC-MS/MS assays for cell wall biosynthesis intermediates would fill a needed gap in the analytical methodology available for antibacterial agent discovery and characterization. An LC-MS/MS assay for several early cell wall intermediates-L-Ala, D-Ala, and D-Ala-D-Ala-has been developed. This method relies on derivatization of bacterial extracts with Marfey's reagent. Marfey's reagent adducts of L-Ala and D-Ala were cleanly separated chromatographically, allowing Marfey's adducts of D-Ala and L-Ala to be separated prior to mass spectrometry (MS) detection and quantitation. The Marfey's adduct of D-Ala-D-Ala was also readily detectable using this same approach. This method shows good linearity (R(2)>0.99), with a lower limit of quantitation of 1 pmol. This assay was demonstrated for characterization of the in vivo effect of cycloserine on Escherichia coli. Cycloserine resulted in a dramatic lowering of both D-Ala and D-Ala-D-Ala levels. Ampicillin had little effect on levels of these three metabolites, consistent with the actions of ampicillin on the later stages of cell wall biosynthesis. These observations indicate that cycloserine inhibits alanine racemase production of D-Ala in E. coli and demonstrates the utility of this assay in directly assessing D-Ala branch targeted antibacterial agents.
