LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens.
Bottom Line: The limited number of validated cellular targets and approaches, along with the increasing amount of antibiotic resistance that is spreading throughout the clinical environment, has prompted us to explore the utility of inhibitors of novel targets and pathways in these resistant organisms, since preexisting target-based resistance should be negligible.Lipid A biosynthesis is an essential process for the formation of lipopolysaccharide, which is a critical component of the Gram-negative outer membrane.In this report, we describe the in vitro and in vivo characterization of novel inhibitors of LpxC, an enzyme whose activity is required for proper lipid A biosynthesis, and demonstrate that our lead compound has the requisite attributes to warrant further consideration as a novel antibiotic.
Affiliation: Discovery Biology, Antibacterials Research Unit, Pfizer Worldwide Research and Development, Groton, Connecticut, USA email@example.com.Show MeSH
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Mentions: Figure 3A shows the predicted structure of the sRNA encoded by wild-type PAO1. In this model, the cytosine 11 bp upstream of the lpxC translational start (filled arrow) is part of a hairpin structure that pairs with the guanine 18 bp upstream of lpxC (open arrow). Changes to this structure when the C-to-A mutation is introduced are shown in Fig. 3B. Given the relatively simplistic nature of this hairpin, we were skeptical about its contribution to regulation of LpxC translation. Unfortunately, our attempts to further characterize this sRNA by restoring the hairpin structure in the mutant through the introduction of a G-to-T mutation 18 bp upstream of lpxC were unsuccessful. Likewise, the cloning of this sRNA into E. coli also did not prove successful, at least not without identifying additional mutations elsewhere within the structure. We were able to demonstrate one key difference between the wild-type and C-to-A mutant strains, however. Although this mutation has historically been regarded as one that impacts translation of LpxC both by us and by others (10), we speculated that the position of this mutation within the sRNA coding region could actually be affecting the transcription or mRNA stability of lpxC. While quantitative RT-PCR (qRT-PCR) experiments using RNA from wild-type PA-1955, PAO397, and their respective C-to-A mutants did not demonstrate any significant differences in mRNA turnover (data not shown), we were encouraged to see a 3-fold increase in lpxC expression in both mutant strains relative to that in their respective parent strains (Fig. 4). While future work is needed to identify any additional components of this regulatory circuit, to our knowledge this is the first report describing a molecular mechanism controlling LpxC production in P. aeruginosa.
Affiliation: Discovery Biology, Antibacterials Research Unit, Pfizer Worldwide Research and Development, Groton, Connecticut, USA firstname.lastname@example.org.