A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

Fan, Jun; Jonge, Boudewijn L. M. de; MacCormack, Kathy; Sriram, Shubha; McLaughlin, Robert E.; Plant, Helen; Preston, Marian; Fleming, Paul R.; Albert, Robert; Foulk, M.A.; Mills, Scott D.

doi:10.1128/aac.03475-14

Cited by 19 publications

(23 citation statements)

References 53 publications

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“…Resistant isolates were passaged on LB agar without selection three times prior to determining the MICs for compounds 1 and 2 to ensure that the isolates were stably resistant mutants. Resistant mutants were then subjected to whole-genome sequencing using an Illumina MiSeq V2 instrument to identify mutations as previously described (25).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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“…However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6,7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9)(10)(11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.…”

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confidence: 99%

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

Osterman

Komarova

Shiryaev

et al. 2016

Antimicrob Agents Chemother

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e In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.T he spread of antibiotic resistance genes among pathogenic bacteria is leading to a gradual decrease in the efficiency of known antibiotics. Substantial efforts have been invested in platforms for new antibiotic development (1). High-throughput screening (HTS) is a major method for the discovery of new chemical scaffolds for drug discovery. However, in the search for new antibiotics, HTS demonstrated low efficiency (for a discussion, see references 2 and 3). Acceleration of the antibiotic development pipeline demands increased efficiency in the identification of mechanisms of action with both HTS of chemical libraries and screening of natural compounds. Ideally, the mechanism of action should be determined while screening for antibacterial activity. One of the major challenges in high-throughput screening is the development of a cost-effective procedure that could maximize information output while concomitantly minimizing the number of pipetting steps and the reagent costs.The most efficient way to reveal the mechanism of action is the application of reporter strains (for a recent review, see reference 4). However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6, 7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9-11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.The majority of antibiotics currently in clinical use target the cell wall, DNA, or protein biosynthesis. For the latter two mechanisms ...

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“…Although a positive signal does not provide definitive MOA assignment, the selective induction of the reporter gene nevertheless affords useful insight into the broad cellular process or metabolic pathway disrupted, irrespective of whether the specific molecular target is known (19,20). Critically, this approach provides a means of biologically profiling compounds with whole-cell activity before extensive medicinal chemistry efforts are undertaken (21,22).…”

mentioning

confidence: 99%

Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery

Naran

Moosa

Barry

et al. 2016

Antimicrob Agents Chemother

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cThe tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis. Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10-to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.

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A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

Cited by 19 publications

References 53 publications

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

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

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery

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