Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.
A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy.


OBJECTIVE
The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.
Department of Bioscience, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Asha S
AS
Nayar
Department of Bioscience, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Thomas J
TJ
Dougherty
Department of Bioscience, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Keith E
KE
Ferguson
Department of Chemistry, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Brett A
BA
Granger
Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
Lisa
L
McWilliams
Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
Clare
C
Stacey
Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
Lindsey J
LJ
Leach
Faculty of Nutritional Sciences, University of Morioka, Takizawa, Iwate, Japan.
Shin-Ichiro
S
Narita
Faculty of Nutritional Sciences, University of Morioka, Takizawa, Iwate, Japan.
Hajime
H
Tokuda
Department of Bioscience, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Alita A
AA
Miller
Department of Chemistry, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Dean G
DG
Brown
Department of Bioscience, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA sarahmmcleod@gmail.com.
Sarah M
SM
McLeod
D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology; D002473:Cell Wall / Q000187:drug effects* / Q000235:genetics; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D010879:Piperazines / Q000302:isolation & purification* / Q000494:pharmacology; D011720:Pyrazoles / Q000302:isolation & purification* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D010879:Piperazines; D011720:Pyrazoles