The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Kenneth N. Kreuzer - One of the best experts on this subject based on the ideXlab platform.
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A Unique Type II Topoisomerase Mutant That Is Hypersensitive to a Broad Range of Cleavage-Inducing Antitumor Agents†
Biochemistry, 2002Co-Authors: Erin K. O'reilly, Kenneth N. KreuzerAbstract:Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target Type II DNA Topoisomerases. Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA. Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in Topoisomerase subunit gp52. Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid [Freudenreich, C. H., et al. (1998) Cancer Res. 58, 1260-1267]. We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid. While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-Type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage. This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors. Mutations that alter drug sensitivities tend to cluster within two specific regions of all Type II Topoisomerases. Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity. We believe that this mutant defines a new category of Type II Topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.
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Bacteriophage T4, a model system for understanding the mechanism of Type II Topoisomerase inhibitors.
Biochimica et Biophysica Acta, 1998Co-Authors: Kenneth N. KreuzerAbstract:Bacteriophage T4 provides a simple model system for analyzing the mechanism of action of antitumor agents that inhibit DNA Topoisomerases. The phage-encoded Type II Topoisomerase is sensitive to many of the same antitumor agents that inhibit mammalian Type II Topoisomerase, including m-AMSA, ellipticines, mitoxantrone and epipodophyllotoxins. Results from the T4 model system provided a convincing demonstration that Topoisomerase is the physiological drug target and strong evidence that the drug-induced cleavage complex is important for cytotoxicity. The detailed molecular steps involved in cytotoxicity, and the mechanism of recombinational repair of inhibitor-induced DNA damage, are currently being analyzed using this model system. Studies with the T4 Topoisomerase have also provided compelling evidence that Topoisomerase inhibitors interact with DNA at the active site of the enzyme, with each class of inhibitor favoring a different subset of cleavage sites based on DNA sequence. Finally, analysis of drug-resistance mutations in the T4 Topoisomerase have implicated certain regions of the protein in drug interaction and provided a strong link between the mechanism of action of the antibacterial quinolones, which inhibit DNA gyrase, and the various antitumor agents, which inhibit mammalian Type II Topoisomerase.
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Localization of an aminoacridine antitumor agent in a Type II Topoisomerase-DNA complex.
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Catherine H. Freudenreich, Kenneth N. KreuzerAbstract:Abstract Type II Topoisomerases are the targets of several classes of chemotherapeutic agents that stabilize an intermediate of the catalytic cycle with the enzyme covalently linked to cleaved DNA. We have used 3-azido-AMSA [4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide], a photo-activatible analog of the inhibitor m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide], to localize the inhibitor binding site in a cleavage complex consisting of an oligonucleotide substrate and the bacteriophage T4 Type II DNA Topoisomerase. Upon photoactivation, the inhibitor covalently attached to the substrate only in the presence of Topoisomerase. Sites of inhibitor attachment were detected by primer-extension analysis and by piperidine-induced cleavage of the covalently modified substrate. 3-Azido-AMSA reacted with bases immediately adjacent to the two phosphodiester bonds cleaved by the enzyme. Therefore, Topoisomerase creates or stabilizes preferential binding sites for the inhibitor precisely at the two sites of DNA cleavage.
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Mutational analysis of a Type II Topoisomerase cleavage site: distinct requirements for enzyme and inhibitors.
The EMBO journal, 1993Co-Authors: Catherine H. Freudenreich, Kenneth N. KreuzerAbstract:Abstract We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 Type II Topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cleavage: four antitumor agents of different classes (an aminoacridine, a substituted anthraquinone, an ellipticine derivative and an epipodophyllotoxin) and one antibacterial quinolone. At eight nucleotide positions flanking the cleavage site, the same preferred bases were found regardless of which inhibitor was present. These preferred bases show dyad symmetry with respect to the cleavage site, indicating that both protomers of the Topoisomerase homodimer interact with DNA in an analogous manner. In addition, we found that the preferred bases on the 5' side of each cleaved phosphodiester bond are highly specific to the inhibitor used in the cleavage reaction. These results strongly suggest that the inhibitors interact directly with the DNA bases at the cleavage site, placing the inhibitor binding site precisely at the site of DNA cleavage.
Anthony Maxwell - One of the best experts on this subject based on the ideXlab platform.
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de novo design of Type II Topoisomerase inhibitors as potential antimicrobial agents targeting a novel binding region
ChemRxiv, 2021Co-Authors: Kyle Orritt, Anthony Maxwell, Juliette Newell, Thomas Germe, Lauren Abbott, Holly Jackson, Benjamin Bury, Martin J Mcphillie, Colin W G FishwickAbstract:By 2050 it is predicted that antimicrobial resistance will be responsible for 10 million global deaths annually, costing the world economy $100 trillion. Clearly, strategies to address this problem are required as bacterial evolution is rendering our current antibiotics ineffective. The discovery of an allosteric binding site on the established antibacterial target DNA gyrase offers a new medicinal chemistry strategy, as this site is distinct from the fluoroquinolone-DNA site binding site. Using in silico molecular design methods, we have designed and synthesised a novel series of biphenyl-based inhibitors inspired by the published thiophene allosteric inhibitor. This series was evaluated in vitro against E. coli DNA gyrase, exhibiting IC50 values in the low micromolar range. The structure-activity relationship reported herein suggests insights to further exploit this allosteric site, offering a pathway to overcome fluoroquinolone resistance.
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the pentapeptide repeat protein mfpa interacts with mycobacterial dna gyrase as a dna t segment mimic
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Lipeng Feng, Julia E A Mundy, Clare E M Stevenson, Lesley A Mitchenall, David M Lawson, Anthony MaxwellAbstract:DNA gyrase, a Type II Topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.
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Exploiting bacterial DNA gyrase as a drug target: Current state and perspectives
Applied Microbiology and Biotechnology, 2011Co-Authors: Frederic Collin, Shantanu Karkare, Anthony MaxwellAbstract:DNA gyrase is a Type II Topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.
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importance of the fourth alpha helix within the cap homology domain of Type II Topoisomerase for dna cleavage site recognition and quinolone action
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: D Strumberg, Anthony Maxwell, John L. Nitiss, Jiaowang Dong, Jerrylaine Walker, Marc C Nicklaus, Kurt W Kohn, Jonathan G Heddle, Siegfried Seeber, Yves PommierAbstract:We report that point mutations causing alteration of the fourth alpha-helix (α4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) Type II Topoisomerases (Ser740Trp, Gln743Pro, and Thr744Pro) change the selection of Type II Topoisomerase-mediated DNA cleavage sites promoted by Ca2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr744Ala substitution had minimal effect on Ca2+- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the α4-helix on Type II Topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser83Trp also changed the DNA cleavage pattern generated by Ca2+ or quinolones. Finally, Thr744Pro substitution in the yeast Type II Topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the α4-helix within the conserved CAP homology domain of Type II Topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the α4-helix are important in determining the activity and possibly the binding of quinolones to the Topoisomerase II-DNA complexes.
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CONVERSION OF DNA GYRASE INTO A CONVENTIONAL Type II Topoisomerase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Sotirios C. Kampranis, Anthony MaxwellAbstract:DNA gyrase is unique among Topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional Type II Topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenoType of a parCts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of Type II Topoisomerases.
David E. Ehmann - One of the best experts on this subject based on the ideXlab platform.
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Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor
Antimicrobial agents and chemotherapy, 2014Co-Authors: Asha S. Nayar, Thomas J. Dougherty, Adam B. Shapiro, Folkert Reck, Jason Thresher, Ning Gao, David E. EhmannAbstract:In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial Type II Topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in Topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in Topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in Topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.
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optimization of physicochemical properties and safety profile of novel bacterial Topoisomerase Type II inhibitors nbtis with activity against pseudomonas aeruginosa
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Folkert Reck, Thomas J. Dougherty, Joseph V. Newman, Sussie Hopkins, Gregory G. Stone, David E. Ehmann, Nikunj Agrawal, Paul Ciaccio, John Mcnulty, Herbert BarthlowAbstract:Type II bacterial Topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial Type II Topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa . Physicochemical properties (p K a and log D ) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa , and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa .
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NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy
Antimicrobial agents and chemotherapy, 2014Co-Authors: Thomas J. Dougherty, Asha S. Nayar, Joseph V. Newman, Sussie Hopkins, Gregory G. Stone, Michele Johnstone, Adam B. Shapiro, Cronin Mark, Folkert Reck, David E. EhmannAbstract:The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial Type II Topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli Topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.
John F Marko - One of the best experts on this subject based on the ideXlab platform.
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Kinetic proofreading can explain the supression of supercoiling of circular DNA molecules by Type-II Topoisomerases
Physical Review E - Statistical Nonlinear and Soft Matter Physics, 2001Co-Authors: Jie Yan, Marcelo O. Magnasco, John F MarkoAbstract:The enzymes that pass DNA through DNA so as to remove entanglements, adenosine-triphosphate-hydrolyzing Type-II Topoisomerases, are able to suppress the probability of self-entanglements (knots) and mutual entanglements (links) between approximately 10 kb plasmids, well below the levels expected, given the assumption that the Topoisomerases pass DNA segments at random by thermal motion. This implies that a 10-nm Type-II Topoisomerase can somehow sense the topology of a large DNA. We previously introduced a "kinetic proofreading" model which supposes the enzyme to require two successive collisions in order to allow exchange of DNA segments, and we showed how it could quantitatively explain the reduction in knotting and linking complexity. Here we show how the same model quantitatively explains the reduced variance of the double-helix linking number (supercoiling) distribution observed experimentally.
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A kinetic proofreading mechanism for disentanglement of DNA by Topoisomerases
Nature, 1999Co-Authors: Jie Yan, Marcelo O. Magnasco, John F MarkoAbstract:Cells must remove all entanglements between their replicated chromosomal DNAs to segregate them during cell division. Entanglement removal is done by ATP-driven enzymes that pass DNA strands through one another, called Type II Topoisomerases. In vitro, some Type II Topoisomerases can reduce entanglements much more than expected, given the assumption that they pass DNA segments through one another in a random way1. These Type II Topoisomerases (of less than 10 nm in diameter) thus use ATP hydrolysis to sense and remove entanglements spread along flexible DNA strands of up to 3,000 nm long. Here we propose a mechanism for this, based on the higher rate of collisions along entangled DNA strands, relative to collision rates on disentangled DNA strands. We show theoretically that if a Type II Topoisomerase requires an initial ‘activating’ collision before a second strand-passing collision, the probability of entanglement may be reduced to experimentally observed levels. This proposed two-collision reaction is similar to ‘kinetic proofreading’ models of molecular recognition2,3.
Thomas J. Dougherty - One of the best experts on this subject based on the ideXlab platform.
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Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor
Antimicrobial agents and chemotherapy, 2014Co-Authors: Asha S. Nayar, Thomas J. Dougherty, Adam B. Shapiro, Folkert Reck, Jason Thresher, Ning Gao, David E. EhmannAbstract:In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial Type II Topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in Topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in Topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in Topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.
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optimization of physicochemical properties and safety profile of novel bacterial Topoisomerase Type II inhibitors nbtis with activity against pseudomonas aeruginosa
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Folkert Reck, Thomas J. Dougherty, Joseph V. Newman, Sussie Hopkins, Gregory G. Stone, David E. Ehmann, Nikunj Agrawal, Paul Ciaccio, John Mcnulty, Herbert BarthlowAbstract:Type II bacterial Topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial Type II Topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa . Physicochemical properties (p K a and log D ) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa , and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa .
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NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy
Antimicrobial agents and chemotherapy, 2014Co-Authors: Thomas J. Dougherty, Asha S. Nayar, Joseph V. Newman, Sussie Hopkins, Gregory G. Stone, Michele Johnstone, Adam B. Shapiro, Cronin Mark, Folkert Reck, David E. EhmannAbstract:The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial Type II Topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli Topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.
