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LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens.

Tomaras AP, McPherson CJ, Kuhn M, Carifa A, Mullins L, George D, Desbonnet C, Eidem TM, Montgomery JI, Brown MF, Reilly U, Miller AA, O'Donnell JP - MBio (2014)

Bottom Line: New antibiotics are needed for the effective treatment of serious infections caused by Gram-negative pathogens, and the responsibility of identifying new drug candidates rests squarely on the shoulders of the infectious disease community.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.

View Article: PubMed Central - PubMed

Affiliation: Discovery Biology, Antibacterials Research Unit, Pfizer Worldwide Research and Development, Groton, Connecticut, USA andrew.tomaras@pfizer.com.

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Related in: MedlinePlus

The sRNA predicted to be encoded in the upstream region of lpxC in P. aeruginosa adopts an altered conformation upon mutation, conferring LpxC-4 resistance. Sequences from the wild-type (A) or C-to-A-mutated (B) variants were analyzed using mfold. The closed arrow indicates the −11 position, while the open arrow indicates the −18 position.
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fig3: The sRNA predicted to be encoded in the upstream region of lpxC in P. aeruginosa adopts an altered conformation upon mutation, conferring LpxC-4 resistance. Sequences from the wild-type (A) or C-to-A-mutated (B) variants were analyzed using mfold. The closed arrow indicates the −11 position, while the open arrow indicates the −18 position.

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.


LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens.

Tomaras AP, McPherson CJ, Kuhn M, Carifa A, Mullins L, George D, Desbonnet C, Eidem TM, Montgomery JI, Brown MF, Reilly U, Miller AA, O'Donnell JP - MBio (2014)

The sRNA predicted to be encoded in the upstream region of lpxC in P. aeruginosa adopts an altered conformation upon mutation, conferring LpxC-4 resistance. Sequences from the wild-type (A) or C-to-A-mutated (B) variants were analyzed using mfold. The closed arrow indicates the −11 position, while the open arrow indicates the −18 position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196226&req=5

fig3: The sRNA predicted to be encoded in the upstream region of lpxC in P. aeruginosa adopts an altered conformation upon mutation, conferring LpxC-4 resistance. Sequences from the wild-type (A) or C-to-A-mutated (B) variants were analyzed using mfold. The closed arrow indicates the −11 position, while the open arrow indicates the −18 position.
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.

Bottom Line: New antibiotics are needed for the effective treatment of serious infections caused by Gram-negative pathogens, and the responsibility of identifying new drug candidates rests squarely on the shoulders of the infectious disease community.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.

View Article: PubMed Central - PubMed

Affiliation: Discovery Biology, Antibacterials Research Unit, Pfizer Worldwide Research and Development, Groton, Connecticut, USA andrew.tomaras@pfizer.com.

Show MeSH
Related in: MedlinePlus