Coli Gyrase Research Articles

Discovery of new Schiff bases of the disalicylic acid scaffold as DNA gyrase and topoisomerase IV inhibitors endowed with antibacterial properties.

DNA gyrase and topoisomerase IV show great potential as targets for antibacterial medicines. In recent decades, various categories of small molecule inhibitors have been identified; however, none have been effective in the market. For the first time, we developed a series of disalicylic acid methylene/Schiff bases hybrids (5a-k) to act as antibacterial agents targeting DNA gyrase and topoisomerase IV. The findings indicated that the new targets 5f-k exhibited significant antibacterial activity against Gram-positive and Gram-negative bacteria, with efficacy ranging from 75% to 115% of the standard ciprofloxacin levels. Compound 5h demonstrated the greatest efficacy compared to the other compounds tested, with minimum inhibitory concentration (MIC) values of 0.030, 0.065, and 0.060μg/mL against S. aureus, E. coli, and P. aeruginosa. 5h had a MIC value of 0.050μg/mL against B. subtilis, which is five times less potent than ciprofloxacin. The inhibitory efficacy of the most potent antibacterial derivatives 5f, 5h, 5i, and 5k against E. coli DNA gyrase was assessed. The tested compounds demonstrated inhibitory effects on E. coli DNA gyrase, with IC50 values ranging from 92 to 112nM. These results indicate that 5f, 5h, 5i, and 5k are more effective than the reference novobiocin, which had an IC50 value of 170nM. Compounds 5f, 5h, 5i, and 5k were subjected to additional assessment against E. coli topoisomerase IV. Compounds 5h and 5i, which have the highest efficacy in inhibiting E. coli gyrase, also demonstrated promising effects on topoisomerase IV. Compounds 5h and 5i exhibit IC50 values of 3.50µM and 5.80 µM, respectively. These results are much lower and more potent than novobiocin's IC50 value of 11µM. Docking studies demonstrate the potential of compound 5h as an effective dual inhibitor against E. coli DNA gyrase and topoisomerase IV, with ADMET analysis indicating promising pharmacokinetic profiles for antibacterial drug development.

Indeno[1,2‐b]pyridin‐5‐one derivatives containing azo groups and their hydrazonal precursors: Synthesis, antimicrobial profile, DNA gyrase binding affinity, and molecular docking

AbstractThe prevalence of germs that are resistant to many antibiotics is rising rapidly the world over. There is a large group of researchers actively looking for better medicines. Here, we designed two series of hydrazonal and indeno[1,2‐b]pyridin‐5‐one bearing hydrazone and azo‐groups to test their antimicrobial activity. Molecular structures of all derivatives were assured based on their spectral data and elemental analyses. Results of the antimicrobial activity of the tested hydrazone and azo compounds showed promising potential for several derivatives. The minimum inhibitory concentrations (MICs) of hydrazones 4a‐h and 6a‐g displayed good antibacterial reactivities with a range of 3.91–250 μg/mL and moderate antifungal activity with a range of 15.6–500 μg/mL. The most promising hydrazone 4f and azo‐6a compounds demonstrated MIC values against Streptococcus faecalis and Escherichia coli equal to 3.91 and 7.81 μg/mL, respectively. Moreover, azo compound 6a showed MIC value equal to 3.91 μg/mL against Enterobacter cloacae species. Additionally, derivative 4f exhibited a significant inhibitory profile against the E. coli gyrase A enzyme (IC50 = 5.53 μg/mL). On the other hand, compound 6a (IC50 14.05 μg/mL) exhibited the lowest DNA gyrase inhibitory activity as compared to compounds 4f and reference standard drug novobiocin, IC50 5.53 and 1.88 μg/mL, respectively. Pharmacokinetic and pharmacodynamic profiles and molecular docking studies for the two most promising molecules 4f and 6a were computed and revealed that both compounds have good ADME profiles and high binding affinity to DNA gyrase binding site.

Novel 2-arylbenzothiazole DNA gyrase inhibitors: Synthesis, antimicrobial evaluation, QSAR and molecular docking studies

A series of new 2-arylbenzothiazole derivatives (4, 5, 6a-j, 7a-i and 8a,b) was synthesized and tested for their antimicrobial activity against different Gram-positive, Gram-negative bacteria and yeast using ciprofloxacin and fluconazole as positive controls for the antibacterial and antifungal activities, respectively. The target compounds showed stronger inhibitory activity against Gram-negative than Gram-positive bacteria. The minimum inhibitory concentration (MIC) values were determined for those compounds showed zone of inhibition ≥ 13 mm. Based on the MIC values for the tested compounds against E. coli, compounds (4, 5, 6c, 6d, 6g, 6i, 6j, 7b, 7c, 7g and 8a) were selected and tested for their E. coli gyrase inhibitory activity. The tested compounds showed moderate inhibitory activity against E. coli gyrase. Compounds 5, 6c, 6i, 6j and 7b displayed high inhibitory activity against E. coli gyrase with IC50 values below 10 µM, however, they were less active than ciprofloxacin (E. coli gyrase IC50 = 1.14 µM). The p-hydroxy-m-methoxy benzothiazole analogue 6c was the most active tested compound (E. coli gyrase IC50 = 4.85 µM). Quantitative structure–activity relationship (QSAR) study was also implemented for the newly synthesized compounds. The QSAR study indicated that the structural feature that governs the anti-microbial activity for the newly synthesized benzothiazole derivatives is their structural hydrophilic-lipophilic balance what agrees with the chemical intuition where this balance governs their cellular absorption and so their antimicrobial activity. Molecular docking showed that the newly synthesized compounds possess the required structural feature for E. coli gyrase B inhibition through interaction with the key amino acids Asp73 and Gly77.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35⁻38 Amino Acids of GyrA.

Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.

Synthesis, antimicrobial activity, and molecular docking study of fluorine-substituted indole-based imidazolines

A series of 2- or 3-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole derivatives were synthesized, characterized, and evaluated for their in vitro antibacterial and antifungal activities. Additionally, the synthesized compounds were docked into the II DNA gyrase B active site, and their predicted binding modes were inspected. Inhibitory activity were tested against two species of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), two species of Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) and two fungi (Candida albicans, Aspergillus niger) using the broth microdilution method. The fluorine-substituted 2-(2-imidazolyl)indole 2b was found to be the most potent antibacterial compound against E. coli and S. aureus strains (MIC value 80 μg/mL). Compounds showed better activity against Gram-positive bacteria compared to Gram-negative bacteria. The docking results predicted that the imidazoline-indole hybrid moiety bind to the active site protein ATP-binding pocket from E. coli and S. aureus with good interaction energy scores. The significant loss of antibacterial activity for some imidazoline-indole analogs could be attributed to several nonoptimal enzyme interactions, including poor hydrogen bonds provided by Asp73 (E. coli gyrase numbering) or Asp81 (S. aureus gyrase numbering) and an associated water molecule.

E. Coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend the gate segment DNA in the process. Bacterial gyrases are a class of type II topoisomerases which introduce negative supercoiling by also tightly wrapping DNA flanking the gate segment before strand passage. To reveal whether topoisomerases can bend or wrap artificially stiffened DNA, E. coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by PCR reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotidetriphosphates. The rates or pauses during relaxation of positive supercoils produced with magnetic tweezers in single molecules of DAP-substituted versus normal DNA distinguished the enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP-dependence consistent with two pathways leading to the strand-passage competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase did not negatively supercoil 35% stiffer DAP DNA most likely by failing to wrap DNA flanking the gate segment before strand passage.

E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.

Ciprofloxacin-Induced Antibacterial Activity Is Attenuated by Phosphodiesterase Inhibitors

BackgroundCiprofloxacin is a commonly used antibiotic for urinary tract infection that interacts with bacterial topoisomerases leading to oxidative radicals generation and bacterial cell death. Phosphodiesterase inhibitors (PDEis), on the other hand, are commonly used drugs for the management of erectile dysfunction. The group includes agents such as sildenafil, vardenafil, and tadalafil. ObjectivesWe investigated whether PDEi could interfere with the antibacterial activity of ciprofloxacin. MethodsPDEis were tested in several reference bacteria, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Acinetobacter baumannii, Proteus mirabilis, and Klebsiella pneumoniae utilizing a standard disc diffusion method and measuring both zones of inhibition and MIC. ResultsResults from both assays indicated that ciprofloxacin demonstrates potent activity against the tested reference bacteria. Additionally, when bacteria were treated with a combination of ciprofloxacin and sildenafil, tadalafil, or vardenafil, the zones of the combination inhibition were significantly reduced, whereas the MIC values were significantly greater than those of ciprofloxacin alone for all tested bacterial strains. In an attempt to examine the mechanism by which PDEis interfere with the action of ciprofloxacin, we utilized the in vitro E coli DNA gyrase cleavage assay. The results showed that PDEi drugs had no effect on ciprofloxacin’s inhibition of E coli gyrase activity. ConclusionsPretreatment of various reference bacterial cells with PDEis largely inhibited the antibacterial activity of ciprofloxacin.

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.

Necroptosis induced by RIPK3 requires MLKL but not Drp1

Necroptosis is a mechanism by which cells can kill themselves that does not require caspase activity or the presence of the pro-apoptotic Bcl-2 family members Bax or Bak. It has been reported that RIPK3 (receptor interacting protein kinase 3) activates MLKL (mixed lineage kinase domain-like) to cause cell death that requires dynamin-related protein 1 (Drp1), because survival was increased in cells depleted of Drp1 or treated with the Drp1 inhibitor mdivi-1. To analyze necroptosis in a system that does not require addition of tumor necrosis factor (TNF), we used a construct that allows RIPK3 to be induced in cells, and then dimerized via an E. coli gyrase domain fused to its carboxyl-terminus, using the dimeric gyrase binding antibiotic coumermycin. We have previously shown elsewhere that RIPK3 dimerized in this manner not only induces necroptosis but also apoptosis, which can be inhibited by the broad-spectrum caspase inhibitor Q-VD-OPh (QVD). In response to RIPK3 dimerization, wild-type mouse embryonic fibroblasts (MEFs) underwent cell death that was reduced but not completely blocked by QVD. In contrast, death upon dimerization of RIPK3 in Mlkl−/− MEFs was completely inhibited with QVD, confirming that MLKL is required for necroptosis. Similar to wild-type MEFs, most Drp1−/− MEFs died when RIPK3 was activated, even in the presence of QVD. Furthermore, overexpression of wild-type MLKL or dominant active mutants of MLKL (Q343A or S345E/S347E) caused death of wild-type and Drp1−/− MEFs that was not inhibited with QVD. These results indicate that necroptosis caused by RIPK3 requires MLKL but not Drp1.

Inhibition of DNA gyrase and DNA topoisomerase IV of Staphylococcus aureus and Escherichia coli by aminocoumarin antibiotics

Aminocoumarin antibiotics are potent inhibitors of bacterial DNA gyrase. We investigated the inhibitory and antibacterial activity of naturally occurring aminocoumarin antibiotics and six structural analogues (novclobiocins) against DNA gyrase and DNA topoisomerase IV from Escherichia coli and Staphylococcus aureus as well as the effect of potassium and sodium glutamate on the activity of these enzymes. The inhibitory concentrations of the aminocoumarins were determined in gyrase supercoiling assays and topoisomerase IV decatenation assays. Both subunits of S. aureus topoisomerase IV were purified as His-Tag proteins in E. coli. The MIC was tested in vivo for the control organisms E. coli ATCC 25922 and S. aureus ATCC 29213. DNA gyrase is the primary target in vitro of all investigated aminocoumarins. With the exception of simocyclinone D8, all other aminocoumarins inhibited S. aureus gyrase on average 6-fold more effectively than E. coli gyrase. Potassium glutamate is essential for the activity of S. aureus gyrase and increases the sensitivity of E. coli gyrase to aminocoumarins ≥ 10-fold. The antibacterial activity of the tested compounds mirrored their relative activities against topoisomerases. The study provides insights about the substituents that are important for the inhibitory activity of aminocoumarins against the target enzymes, which will facilitate the rational design of improved antibiotics.

Structure of the E. Coli Gyrase DNA Binding and Cleavage Core Reveals A Unique Domain

DNA topoisomerases are essential enzymes that sustain chromosome supercoiling homeostasis in all forms of life. DNA gyrase, a heterotetrameric type IIA topoisomerase, has the unique ability to introduce negative supercoils into DNA, helping maintain bacterial genomes in a compact, underwound state. Though all gyrase orthologs use a set of homologous domains and a central “two-gate” mechanism for passing one DNA segment through another, they also exhibit critical family-specific differences. For example, the metal- and DNA-binding TOPRIM domain of gyrases found in many gamma- and beta-proteobacteria contains a 170-amino acid insertion of unknown function. We have solved the crystal structure of the E.coli gyrase DNA binding and cleavage core, visualizing this insertion for the first time. Biochemical analyses of a structure-guided deletion mutant lacking this region reveal that it may help coordinate the activities of gyrase's distal ATPase and DNA binding gates.

First functional characterization of a singly expressed bacterial type II topoisomerase: The enzyme from Mycobacterium tuberculosis

Genome deciphering revealed that Mycobacterium tuberculosis encodes a single type II topoisomerase contrary to common bacteria harboring two type II topoisomerases (DNA gyrase and topoisomerase IV). Functions of the M. tuberculosis type II topoisomerase were explored after cloning and expressing the subunits encoding genes in Escherichia coli. M. tuberculosis type II topoisomerase supercoiled relaxed pBR322 with a specific activity close to that of DNA gyrases of common bacteria whereas it exhibited DNA relaxation and formation of cleavable complexes with activities significantly higher than other DNA gyrases. Intermolecular passage activity evaluated by the decatenation of kinetoplast DNA was 25-fold lower than that of the topoisomerase IV from Streptococcus pneumoniae, but was markedly higher than that of the E. coli gyrase. Overall, the type II topoisomerase of M. tuberculosis exhibits classical polyvalent activities of DNA gyrase for supercoiling but enhanced relaxation, cleavage, and decatenation activities.

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

The Topodynamics of Incision of UV-irradiated Covalently Closed DNA by the Escherichia coli Uvr(A)BC Endonuclease

The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide excision repair of a large variety of DNA damages. The damage recognition and incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent DNA helix-tracking activity associated with the UvrA2B1 complex. The latter activity leads to the generation of highly positively supercoiled DNA in the presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can contribute to the above reaction by abolishing the accumulation of highly positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision. Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to incise highly positively supercoiled uvDNA results from the failure of the formation of UvrAB-dependent obligatory intermediates associated with the DNA conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV endonuclease efficiently incises uvDNA regardless of its topological state. The in vitro topodynamic system proposed in this study may provide a simple model for studying a topological aspect of nucleotide excision repair and its interaction with other DNA topology-related processes in E. coli.

Transcriptional block caused by a negative supercoiling induced structural change in an alternating CG sequence

Using supercoiled plasmids containing a (CG) 16 sequence downstream of a promoter, it is shown that purified E. coli RNA polymerase can transcribe through the sequence when it is in the B helical form. However, the polymerase together with its nascent transcript is blocked at the boundary of the CG sequence proximal to the promoter when the template is negatively supercoiled to flip the CG sequence to the left-handed Z-form. S1 nuclease mapping of in vivo transcripts from an E. coli gyrase temperature-sensitive mutant harboring the plasmids indicates that the bulk of the transcripts at either permissive or nonpermissive temperatures can proceed through the CG sequence, suggesting that the sequence is normally in the B helical form in vivo. The almost total blockage of transcription in vitro by the (CG) 16 sequence in a highly negatively supercoiled DNA is not observed for a d(CA) 21 · d(TG) 21 insert.