Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

Kühl, Alexander; Svenstrup, Niels; Ladel, Christoph; Otteneder, Michael B.; Binas, Annegret; Schiffer, Guido; Brands, Michael; Lampe, Thomas; Ziegelbauer, Karl; Rübsamen‐Waigmann, Helga; Haebich, Dieter; Ehlert, Kerstin

doi:10.1128/aac.49.3.987-995.2005

Cited by 24 publications

(20 citation statements)

References 27 publications

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“…The yorB marker has recently been described as being strongly induced by quinolones (DNA gyrase/topoisomerase IV inhibitors) (19). We found that many more compounds induced the reporter: the DNA-alkylating agent mitomycin C, the folate biosynthesis inhibitor trimethoprim, and the recently reported novel inhibitors of the DNA polymerase IIIC (anilinouracils) (24) and the bacterial NAD ϩ -dependent DNA ligase (pyridochromanones) (6) as strong inducers (Ͼ500%). In addition, the DNA gyrase inhibitor novobiocin was detected, even though the signal was only raised to 250%.…”

Section: Resultsmentioning

confidence: 76%

“…3 (6), moiramide B, moxifloxacin, polymerase IIIC inhibitor compound no. 1 (24), and streptovaricin (Bayer); ramoplanin (Biosearch Italia, Geranzano, Italy); trovafloxacin (Pfizer); and actinomycin D, actinonin, azaserine, azithromycin, cefoxitin, cerulenin, chloramphenicol, doxycycline, erythromycin, ethidium bromide, fusidic acid, gentamicin, kanamycin, mersacidin, methicillin, monensin, nalidixic acid, netropsin, N-ethyl maleimide, nisin, novobiocin, mitomycin C, oxacillin, polymyxin B, puromycin, triclosan, trimethoprim, and tunicamycin (Sigma-Aldrich). The Bayer collection of pure natural products consisted of approximately 14,000 individual substances which were each characterized by nuclear magnetic resonance spectroscopy.…”

Section: Methodsmentioning

confidence: 99%

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Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

Urban

Eckermann

Fast

et al. 2007

Appl Environ Microbiol

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113

112

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Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of <25 g/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

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Section: Resultsmentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

Urban

Eckermann

Fast

et al. 2007

Appl Environ Microbiol

Self Cite

113

112

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“…The diverse PHP family is associated with a range of hydrolase activities, with many PHPs still uncharacterized. Like other PHP structures, the PolC PHP exhibits a distorted (␤␣) 7 -barrel and coordinates up to 3 metals and a phosphate (Fig. S3).…”

Section: Polc Php Domain Has a Metal-binding Cluster Yet Lacks Catalyticmentioning

confidence: 99%

“…The major C family replicative polymerases are DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC (PolIIIC), found primarily in Gram-positive bacteria (4). Apoenzyme crystal structures of DnaE have revealed surprising structural differences in the catalytic center of the enzyme compared with B family polymerases, suggesting a separate evolutionary origin for the C family (5, 6).As the core component of the replicative polymerase complex in Gram-positive pathogens such as Staphylococcus aureus, PolC has received considerable attention as a potential target for antibacterial drug discovery (7,8). No currently marketed antibiotics target the central replication apparatus, making PolC a novel target for antibacterial development.…”

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confidence: 99%

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Structure of PolC reveals unique DNA binding and fidelity determinants

Evans

Davies²,

Bullard

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

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PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-Å resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique ␤-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.DNA polymerase III ͉ DNA replication ͉ Gram-positive polymerase ͉ polymerase and histidinol phosphatase (PHP) ͉ ternary complex D NA polymerases are the enzymes responsible for DNA synthesis. Cellular organisms typically use multiple DNA polymerase types. The ''replicative'' polymerase performs the bulk of genome duplication, whereas various specialty polymerases repair damaged DNA and resolve Okazaki fragments. Across every kingdom of life, replicative polymerases exhibit certain hallmarks such as high fidelity, speed, and processivity (1). Polymerase holoenzyme accessory proteins play an integral role in achieving the extraordinary efficiency and accuracy of the replicative polymerase complex. These include a ''sliding clamp'' that encircles the DNA and increases processivity (2).Bacterial replicative polymerases comprise the C family of DNA polymerases (3) and differ significantly from the replicative polymerases of eukaryotes, bacteriophage, and archaea, which belong to the B family. The major C family replicative polymerases are DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC (PolIIIC), found primarily in Gram-positive bacteria (4). Apoenzyme crystal structures of DnaE have revealed surprising structural differences in the catalytic center of the enzyme compared with B family polymerases, suggesting a separate evolutionary origin for the C family (5, 6).As the core component of the replicative polymerase complex in Gram-positive pathogens such as Staphylococcus aureus, PolC has received considerable attention as a potential target for antibacterial drug discovery (7,8). No currently marketed antibiotics target the central replication apparatus, making PolC a novel target for antibacterial development. Gram-negative and Gram-positive bacteria are separated by Ͼ1 billion years of evolution, and PolC and DnaE share Ͻ20% sequence identity; PolC is further differentiated from DnaE by domain rearrangements and by the presence of an ...

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Polypharmacology as an Emerging Trend in Antibacterial Discovery

Silver¹

2012

Polypharmacology in Drug Discovery

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Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

Cited by 24 publications

References 27 publications

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

Structure of PolC reveals unique DNA binding and fidelity determinants

Polypharmacology as an Emerging Trend in Antibacterial Discovery

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