Fluoroquinolone-Gyrase-DNA Complexes

Mustaev, Arkady; Malik, Muhammad Abdul; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M.; Hiasa, Hiroshi; Marks, Kevin M.; Kerns, Robert J.; Berger, James M.; Drlica, Karl

doi:10.1074/jbc.m113.529164

Cited by 135 publications

(108 citation statements)

References 53 publications

(30 reference statements)

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“…From this observation, it can be hypothesized that the mutation pattern observed in F. novicida is not based on the in vitro selection protocol but is likely to be observed in vivo. The preponderant role of GyrB in FQ resistance highlighted here fits well with the recently proposed inverted binding mode of FQs to gyrase-DNA complexes (27). Using Mycobacterium smegmatis as a model, Mustaev et al (27) indeed proposed a role for the GyrB E466 or K447 residue as an alternative way to ensure stabilization of the drug-enzyme-DNA complex known to be mediated through the magnesium-water bridge with GyrA residues 83 and 87.…”

Section: Discussionsupporting

confidence: 55%

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Caspar

Siebert

Sutera

et al. 2017

Antimicrob Agents Chemother

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Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC 50 ) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

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Section: Discussionsupporting

confidence: 55%

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Caspar

Siebert

Sutera

et al. 2017

Antimicrob Agents Chemother

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“…Much attention has already been paid to modifying C7 ring groups, with clear success (23,49,56); it seems likely that these improvements could be further capitalized upon by using longer linkers at the C7 position and allowing substituents to reach and contact side chains of amino acids such as Arg482, Thr500, or Glu501 in a more specific manner. In such an approach, it might be possible to pin a fluoroquinolone in the gyrase drug-binding pocket, using a water/magnesium-ion bridge from GyrA to anchor the keto-acid group at one end of the drug and specific contacts from GyrB to C7 features at the other; indeed, cross-links can be formed between the C7 end of ciprofloxacin and GyrB-Glu466 of E. coli gyrase (equivalent to Mtb GyrB-Glu501) (50). This feature may be important for designing not only more active fluoroquinolones but also more active quinazolinediones (57).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure and stability of gyrase–fluoroquinolone cleaved complexes from Mycobacterium tuberculosis

Blower

Williamson

Kerns

et al. 2016

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

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177

167

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Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and in 2013 accounted for 1.5 million deaths. Fluoroquinolone antibacterials, which target DNA gyrase, are critical agents used to halt the progression from multidrug-resistant tuberculosis to extensively resistant disease; however, fluoroquinolone resistance is emerging and new ways to bypass resistance are required. To better explain known differences in fluoroquinolone action, the crystal structures of the WT Mtb DNA gyrase cleavage core and a fluoroquinolone-sensitized mutant were determined in complex with DNA and five fluoroquinolones. The structures, ranging from 2.4-to 2.6-Å resolution, show that the intrinsically low susceptibility of Mtb to fluoroquinolones correlates with a reduction in contacts to the water shell of an associated magnesium ion, which bridges fluoroquinolone-gyrase interactions. Surprisingly, the structural data revealed few differences in fluoroquinoloneenzyme contacts from drugs that have very different activities against Mtb. By contrast, a stability assay using purified components showed a clear relationship between ternary complex reversibility and inhibitory activities reported with cultured cells. Collectively, our data indicate that the stability of fluoroquinolone/DNA interactions is a major determinant of fluoroquinolone activity and that moieties that have been appended to the C7 position of different quinolone scaffolds do not take advantage of specific contacts that might be made with the enzyme. These concepts point to new approaches for developing quinolone-class compounds that have increased potency against Mtb and the ability to overcome resistance.Mycobacterium tuberculosis | antibiotic resistance | fluoroquinolone | gyrase | complex stability

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“…The multiple structures of topoisomerase proteins bound to fluoroquinolones (16,20,40,42) reveal minimal differences in the interaction of these residues with the DNA bound to fluoroquinolones versus the NBTI complex, although the conformation of the DNA strand is slightly different (Fig. 2A).…”

Section: ϫ8mentioning

confidence: 99%

Insights into the Mechanism of Inhibition of Novel Bacterial Topoisomerase Inhibitors from Characterization of Resistant Mutants of Staphylococcus aureus

Lahiri

Kutschke

McCormack

et al. 2015

Antimicrob Agents Chemother

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The type II topoisomerases DNA gyrase and topoisomerase IV are clinically validated bacterial targets that catalyze the modulation of DNA topology that is vital to DNA replication, repair, and decatenation. Increasing resistance to fluoroquinolones, which trap the topoisomerase-DNA complex, has led to significant efforts in the discovery of novel inhibitors of these targets. AZ6142 is a member of the class of novel bacterial topoisomerase inhibitors (NBTIs) that utilizes a distinct mechanism to trap the protein-DNA complex. AZ6142 has very potent activity against Gram-positive organisms, including Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. In this study, we determined the frequencies of resistance to AZ6142 and other representative NBTI compounds in S. aureus and S. pneumoniae. The frequencies of selection of resistant mutants at 4؋ the MIC were 1.7 ؋ 10 ؊8 for S. aureus and <5.5 ؋ 10 ؊10 for S. pneumoniae. To improve our understanding of the NBTI mechanism of inhibition, the resistant S. aureus mutants were characterized and 20 unique substitutions in the topoisomerase subunits were identified. Many of these substitutions were located outside the NBTI binding pocket and impact the susceptibility of AZ6142, resulting in a 4-to 32-fold elevation in the MIC over the wild-type parent strain. Data on cross-resistance with other NBTIs and fluoroquinolones enabled the differentiation of scaffold-specific changes from compound-specific variations. Our results suggest that AZ6142 inhibits both type II topoisomerases in S. aureus but that DNA gyrase is the primary target. Further, the genotype of the resistant mutants suggests that domain conformations and DNA interactions may uniquely impact NBTIs compared to fluoroquinolones.

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Fluoroquinolone-Gyrase-DNA Complexes

Cited by 135 publications

References 53 publications

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Crystal structure and stability of gyrase–fluoroquinolone cleaved complexes from Mycobacterium tuberculosis

Insights into the Mechanism of Inhibition of Novel Bacterial Topoisomerase Inhibitors from Characterization of Resistant Mutants of Staphylococcus aureus

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