Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

Abstract

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. IMPORTANCEThe 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.

Redefining the essential trafficking pathway for outer membrane lipoproteins

“…S3). Accordingly, we also observed that cpxA24 Δlpp ΔrcsB cells remained sensitive to "compound 2," a pyrazole inhibitor of LolC and LolE (Table S1) (44). The minimum inhibitory concentration (MIC) of compound 2 was significantly lower in the ΔlolA and ΔlolB derivatives of cpxA24 Δlpp ΔrcsB.…”

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

“…Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10)(11)(12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).…”

Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

“…Strains exist with efflux pumps knocked out either individually or in combination, and the relative contribution of each system to the susceptibility to major classes of antimicrobials has been defined (15). For the most part, tolC is the major contributor to efflux in E. coli, and knockout of this gene is often used to assess whether novel compounds lack cellular activity due to efflux (16)(17)(18)(19)(20)(21)(22)(23)(24). Similarly, mutations in several genesincluding lpxC (25-27), lptD (28, 29), and lptE (30, 31)-leading to increased permeability of E. coli have been described, and strains harboring such lesions are often used to assess the effect of increased cellular penetration on the bioactivity of molecules (32-37).…”