The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE), Part II: Development of inhibitors with broad spectrum,Gram-negative antibacterial activity

The structurally related bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as prime candidates for the development of broad spectrum antibacterial agents. However, GyrB/ParE targeting antibacterials with spectrum that encompasses robust Gram-negative pathogens have not yet been reported. Using structure-based inhibitor design, we optimized a novel pyrrolopyrimidine inhibitor series with potent, dual targeting activity against GyrB and ParE. Compounds were discovered with broad antibacterial spectrum, including activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. Herein we describe the SAR of the pyrrolopyrimidine series as it relates to key structural and electronic features necessary for Gram-negative antibacterial activity.     

DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: Synthesis and antibacterial activity

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.     

Synthesis,evaluation and molecular docking studies of amino acid derived N-glycoconjugates as antibacterial agents

Six amino acid derived N-glycoconjugates of d-glucose were synthesized, characterized and tested for antibacterial activity against G(+)ve (Bacillus cereus) as well as G(−)ve (Escherichia coli and Klebsiella pneumoniae) bacterial strains. All the tested compounds exhibited moderate to good antibacterial activity against these bacterial strains. The results were compared with the antibacterial activity of standard drug Chloramphenicol, where results of A5 (Tryptophan derived glycoconjugates) against E. coli and A4 (Isoleucine derived glycoconjugates) against K. pneumoniae bacterial strains are comparable with the standard drug molecule. In silico docking studies were also performed in order to understand the mode of action and binding interactions of these molecules. The docking studies revealed that, occupation of compound A5 at the ATP binding site of subunit GyrB (DNA gyrase, PDB ID: 3TTZ) via hydrophobic and hydrogen bonding interactions may be the reason for its significant in vitro antibacterial activity.     

A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA. Absent in humans, gyrase is a successful target for antibacterial drugs. However, increasing drug resistance is a serious problem and new agents are urgently needed. The naturally-produced Escherichia coli toxin CcdB has been shown to target gyrase by what is predicted to be a novel mechanism. CcdB has been previously shown to stabilize the gyrase ‘cleavage complex’, but it has not been shown to inhibit the catalytic reactions of gyrase. We present data showing that CcdB does indeed inhibit the catalytic reactions of gyrase by stabilization of the cleavage complex and that the GyrA C-terminal DNA-wrapping domain and the GyrB N-terminal ATPase domain are dispensable for CcdB's action. We further investigate the role of specific GyrA residues in the action of CcdB by site-directed mutagenesis; these data corroborate a model for CcdB action based on a recent crystal structure of a CcdB–GyrA fragment complex. From this work, we are now able to present a model for CcdB action that explains all previous observations relating to CcdB–gyrase interaction. CcdB action requires a conformation of gyrase that is only revealed when DNA strand passage is taking place.     

Binding of two DNA molecules by type II topoisomerases for decatenation

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.     

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.     

Discovery of N-quinazolinone-4-hydroxy-2-quinolone-3-carboxamides as DNA gyrase B-targeted antibacterial agents

Emerging drug resistance is generating an urgent need for novel and effective antibiotics. A promising target that has not yet been addressed by approved antibiotics is the bacterial DNA gyrase subunit B (GyrB), and GyrB inhibitors could be effective against drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA). Here, we used the 4-hydroxy-2-quinolone fragment to search the Specs database of purchasable compounds for potential inhibitors of GyrB and identified AG-690/11765367, or f1, as a novel and potent inhibitor of the target protein (IC₅₀: 1.21 µM). Structural modification was used to further identify two more potent GyrB inhibitors: f4 (IC₅₀: 0.31 µM) and f14 (IC₅₀: 0.28 µM). Additional experiments indicated that compound f1 is more potent than the others in terms of antibacterial activity against MRSA (MICs: 4–8 µg/mL), non-toxic to HUVEC and HepG2 (CC₅₀: approximately 50 µM), and metabolically stable (t_1/2: > 372.8 min for plasma; 24.5 min for liver microsomes). In summary, this study showed that the discovered N-quinazolinone-4-hydroxy-2-quinolone-3-carboxamides are novel GyrB-targeted antibacterial agents; compound f1 is promising for further development.     

Identification of an ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate as a catalytic inhibitor of DNA gyrase

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC₅₀ of 3.5 μM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.     

A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72 415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.     

Expression inEscherichia coliof Y5-Mutant and N-terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions.     

Probing the binding of the coumarin drugs using peptide fragments of DNA gyrase B protein.

Bacterial DNA gyrase, has been identified as the target of several antibacterial agents, including the coumarin drugs. The coumarins inhibit the gyrase action by competitive binding to the ATP-binding site of DNA gyrase B (GyrB) protein. The high in vitro inhibitory potency of coumarins against DNA gyrase reactions has raised interest in studies on coumarin-gyrase interactions. In this context, a series of low-molecular weight peptides, including the coumarin resistance-determining region of subunit B of Escherichia coli gyrase, has been designed and synthesized. The first peptide model was built using the natural fragment 131-146 of GyrB and was able to bind to novobiocin (K(a) = 1.8 +/- 0.2 x 10(5)/m) and ATP (K(a) = 1.9 +/- 0.4 x 10(3)/m). To build the other sequences, changes in the Arg(136) residue were introduced so that the binding to the drug was progressively reduced with the hydrophobicity of this residue (K(a) = 1.3 +/- 0.1 x 10(5)/m and 1.0 +/- 0.2 x 10(5)/m for Ser and His, respectively). No binding was observed for the change Arg(136) to Leu. In contrast, the binding to ATP was not altered, independently of the changes promoted. On the contrary, for peptide-coumarin and peptide-ATP complexes, Mg(2+) appears to modulate the binding process. Our results demonstrate the crucial role of Arg(136) residue for the stability of coumarin-gyrase complex as well as suggest a different binding site for ATP and in both cases the interactions are mediated by magnesium ions.     

A high-throughput fluorescence polarization assay for inhibitors of gyrase B

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.     

Replacement of cardiotoxic aminopiperidine linker with piperazine moiety reduces cardiotoxicity? Mycobacterium tuberculosis novel bacterial topoisomerase inhibitors

Recently numerous non-fluoroquinolone-based bacterial type II topoisomerase inhibitors from both the GyrA and GyrB classes have been reported as antibacterial agents. Inhibitors of the GyrA class include aminopiperidine-based novel bacterial type II topoisomerase inhibitors (NBTIs). However, inhibition of the cardiac ion channel remains a serious liability for the aminopiperidine based NBTIs. In this paper we replaced central aminopiperidine linker with piperazine moiety and tested for its biological activity. We developed a series of twenty four compounds with a piperazine linker 1-(2-(piperazin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one, by following a multistep protocol. Among them compound 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-N-(4-nitrophenyl)piperazine-1-carboxamide (11) was the most promising inhibitor with Mycobacterium tuberculosis (MTB) DNA gyrase enzyme supercoiling IC₅₀ of 0.29 ± 0.22 μM, with a good MTB MIC of 3.45 μM. These kind of compounds retains good potency and showed reduced cardiotoxicity compared to aminopiperidines.     

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.     

Fluoroquinolone-Gyrase-DNA Complexes: TWO MODES OF DRUG BINDING*

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys⁴⁶⁶ gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly⁸¹ and GyrB-Glu⁴⁶⁶ residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.     

Biophysical characterization of an indolinone inhibitor in the ATP-binding site of DNA gyrase

Fighting bacterial resistance is a challenging task in the field of medicinal chemistry. DNA gyrase represents a validated antibacterial target and has drawn much interest in recent years. By a structure-based approach we have previously discovered compound 1, an indolinone derivative, possessing inhibitory activity against DNA gyrase. In the present paper, a detailed biophysical characterization of this inhibitor is described. Using mass spectrometry, NMR spectroscopy, and fluorescence experiments we have demonstrated that compound 1 binds reversibly to the ATP-binding site of the 24 kDa N-terminal fragment of DNA gyrase B from Escherichia coli (GyrB24) with low micromolar affinity. Based on these data, a plausible molecular model of compound 1 in the active site of GyrB24 was constructed. The predicted binding mode explains the competitive inhibitory mechanism with respect to ATP and forms a useful basis for further development of potent DNA gyrase inhibitors.     

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg²⁺ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K_0.5 and k_cat of 0.68 mM and 0.39 s^–1, respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.     

Discovery and development of kibdelomycin,a new class of broad-spectrum antibiotics targeting the clinically proven bacterial type II topoisomerase

Kibdelomycin is a complex novel antibiotic, discovered by applying a highly sophisticated chemical-genetic Staphylococcus aureus Fitness Test (SaFT) approach, that inhibits the clinically established bacterial targets, gyrase and topoisomerase IV. It exhibits broad-spectrum antibacterial activity against aerobic bacteria including MRSA and Acinetobacter baumannii. It is slowly bactericidal and has a low frequency of resistance. In an anaerobic environment, it exhibits narrow-spectrum activity and inhibits the growth of gut bacteria Clostridium difficile (MIC 0.125 μg/mL) without affecting the growth of commensal Gram-negative organisms particularly, Bacteroides sp. It is highly efficacious in the hamster model of C. difficile infection providing 100% protection at >6 mg/kg and 80% protection at 1.56 mg/kg by oral dosing without systemic exposure. X-ray co-crystal structures of kibdelomycin bound to GyrB and ParE showed a unique dual arm ‘U shaped’ multisite binding never encountered with any other gyrase inhibitors. Kibdelomycin is poised for preclinical development for C. difficile treatment, and most importantly, the co-crystal structures of kibdelomycin provide unique insight for structure-guided structure modification, which could lead to better broader-spectrum systemic antibiotic potentially covering many ESKAPE pathogens.