A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

Optimization of TopoIV Potency, ADMET Properties, and hERG Inhibition of 5-Amino-1,3-dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Identification of a Lead with In Vivo Efficacy against MRSA.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

Design, Synthesis and Biological Evaluation of Novel Heterocyclic Fluoroquinolone Citrate Conjugates as Potential Inhibitors of Topoisomerase IV: A Computational Molecular Modeling Study.

BACKGROUND & OBJECTIVE: A facile and efficient method for the synthesis of novel derivatives of FQ citrate conjugates with 1,2,4-triazoles and 1,3,4-oxadiazole scaffolds 8-11 using conventional, as well as microwave irradiation methods, was reported. Based on these original building blocks, the new derivatives of 3, 7-disubstituted fluoroquinolones bearing the oxadiazolyl-triazole groups were obtained. These invaluable derivatives are of great interest in medicinal and pharmaceutical studies because of their important biological properties. METHODS: All the reactions were examined under conventional as well as microwave mediated conditions. The structures of obtained compounds were confirmed by 1H NMR, 13C NMR, IR HRMS spectroscopy, and elemental analysis. The antibacterial and antifungal activities of these compounds were screened against Gram-positive, Gram-negative bacteria, and fungal stains by the agar well diffusion method. Cytotoxic assay of the title compounds was evaluated against cervical carcinoma cell line (HeLa) by using the MTT assay. The crystal structure of the Quinolone-DNA cleavage complex of type IV topoisomerase from S. pneumoniae (PDB ID: 3RAE) complex was obtained from the Protein Database (PDB, http:// www.rcsb.org). Molecular properties prediction-drug likeness was studied by Molinspiration and Molsoft software, while lipophilicity and solubility parameters were studied using the Osiris program. RESULTS: A novel approach for the synthesis of benzylthio-1,2,4-triazole and 1,3,4-oxadiazoles core with regioisomeric norfloxacin citrate conjugates was developed. Among the title compounds, 11b, 10a reveal pronounced activity against S. pneumoniae with minimum inhibitory concentrations of 0.89, 0.96 mg/mL and MBCs of 2.95, 2.80 mg/mL, respectively. Minimum Fungicidal Concentration (MFC) has been determined for each compound against two fungal strains. Compound 11b showed maximum anti-cancer activity against HeLa cell line with IC50 value 11.3 ± 0.41 comparable to standard drug DXN. For binding mode, active site residues and docking energies (ΔG =-7.9 Kcal/mol) for ligand 9b exhibited the highest hydrogen bonding (3.59274 A˚), Pi- Alkyl (5.14468 A˚) interactions with amino acid LEU479 of 3RAE protein. The compounds following the Lipinski 'Rule of five' were synthesized for antimicrobial and anti-cancer screening as oral bioavailable drugs/leads. Maximum drug likeness model score 1.52, 1.41 was found for compounds 10d, 11b. CONCLUSION: The present work, through simple synthetic approaches, led to the development of novel hybrids of fluoroquinolone containing citrate-triazole-oxadiazole pharmacophores that exhibited remarkable biological activities against different microorganisms and cell lines. The compounds showed suitable druglike properties and are expected to present good bioavailability profile. An efficient combination of molecular modeling and biological activity provided an insight into QSAR guidelines that could aid in further development and optimization of the norfloxacin derivatives.

Discovery of Pyrido[2,3-b]indole Derivatives with Gram-Negative Activity Targeting Both DNA Gyrase and Topoisomerase IV.

The rise of multidrug resistant (MDR) Gram-negative (GN) pathogens and the decline of available antibiotics that can effectively treat these severe infections are a major threat to modern medicine. Developing novel antibiotics against MDR GN pathogens is particularly difficult as compounds have to permeate the GN double membrane, which has very different physicochemical properties, and have to circumvent a plethora of resistance mechanisms such as multiple efflux pumps and target modifications. The bacterial type II topoisomerases DNA gyrase (GyrA2B2) and Topoisomerase IV (ParC2E2) are highly conserved targets across all bacterial species and validated in the clinic by the fluoroquinolones. Dual inhibitors targeting the ATPase domains (GyrB/ParE) of type II topoisomerases can overcome target-based fluoroquinolone resistance. However, few ATPase inhibitors are active against GN pathogens. In this study, we demonstrated a successful strategy to convert a 2-carboxamide substituted azaindole chemical scaffold with only Gram-positive (GP) activity into a novel series with also potent activity against a range of MDR GN pathogens. By systematically fine-tuning the many physicochemical properties, we identified lead compounds such as 17r with a balanced profile showing potent GN activity, high aqueous solubility, and desirable PK features. Moreover, we showed the bactericidal efficacy of 17r using a neutropenic mouse thigh infection model.

Trapping of the transport-segment DNA by the ATPase domains of a type II topoisomerase.

Type II topoisomerases alter DNA topology to control DNA supercoiling and chromosome segregation and are targets of clinically important anti-infective and anticancer therapeutics. They act as ATP-operated clamps to trap a DNA helix and transport it through a transient break in a second DNA. Here, we present the first X-ray crystal structure solved at 2.83 Å of a closed clamp complete with trapped T-segment DNA obtained by co-crystallizing the ATPase domain of S. pneumoniae topoisomerase IV with a nonhydrolyzable ATP analogue and 14-mer duplex DNA. The ATPase dimer forms a 22 Å protein hole occupied by the kinked DNA bound asymmetrically through positively charged residues lining the hole, and whose mutagenesis impacts the DNA decatenation, DNA relaxation and DNA-dependent ATPase activities of topo IV. These results and a side-bound DNA-ParE structure help explain how the T-segment DNA is captured and transported by a type II topoisomerase, and reveal a new enzyme-DNA interface for drug discovery.

Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC90=0.125µg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC50 of 85.9µM and a favorable profile in the anesthetized guinea pig model.

Molecular Markers of Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus SCCmec IV Presenting Different Genetic Backgrounds.

Methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmec type IV has emerged in hospitals worldwide. The aim of this study was to evaluate phenotypic and molecular characteristics of antimicrobial resistance in MRSA SCCmec IV isolates, presenting different genetic backgrounds, isolated from hospitals in Rio de Janeiro. The antimicrobial resistance of 128 S. aureus type IV isolates from 11 hospitals was characterized by the disk diffusion test and minimum inhibitory concentration (MIC) test. Mutations in parC gene, which encodes ciprofloxacin resistance, and genes associated with macrolide-lincosamide-streptogramin B (MLSb) resistance were also investigated. MRSA isolates belonging to USA400/ST1 (60 isolates), USA800/ST5 (40), USA1100/ST30 (13), and other 11 (15) lineages were mainly resistant to erythromycin (68%), ciprofloxacin (56%), and clindamycin (50%). The highest antimicrobial resistance rates were found among USA400 isolates (p < 0.05). The majority of them (90%) carried only the erm(C) gene and mainly presented two mutation types in the parC gene. The msr(A) gene was most frequently found among USA800 isolates (p < 0.05). Among MRSA type IV isolates from Rio de Janeiro hospitals, multiresistance, including mutations in parC gene, was associated to the USA400/ST1, while the msr(A) gene was associated with USA800/ST5 isolates, highlighting that these lineages could have more potential to persist in a hospital environment.

Fragment-based discovery of DNA gyrase inhibitors targeting the ATPase subunit of GyrB.

Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.

Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor.

Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.

Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate.

Activity of quinolone CP-115,955 against bacterial and human type II topoisomerases is mediated by different interactions.

CP-115,955 is a quinolone with a 4-hydroxyphenyl at C7 that displays high activity against both bacterial and human type II topoisomerases. To determine the basis for quinolone cross-reactivity between bacterial and human enzymes, the activity of CP-115,955 and a series of related quinolones and quinazolinediones against Bacillus anthracis topoisomerase IV and human topoisomerase IIα was analyzed. Results indicate that the activity of CP-115,955 against the bacterial and human enzymes is mediated by different interactions. On the basis of the decreased activity of quinazolinediones against wild-type and resistant mutant topoisomerase IV and the low activity of quinolones against resistant mutant enzymes, it appears that the primary interaction of CP-115,955 with the bacterial system is mediated through the C3/C4 keto acid and the water-metal ion bridge. In contrast, the drug interacts with the human enzyme primarily through the C7 4-hydroxyphenyl ring and has no requirement for a substituent at C8 in order to attain high activity. Despite the fact that the human type II enzyme is unable to utilize the water-metal ion bridge, quinolones in the CP-115,955 series display higher activity against topoisomerase IIα in vitro and in cultured human cells than the corresponding quinazolinediones. Thus, quinolones may be a viable platform for the development of novel drugs with anticancer potential.

Biological evaluation of benzothiazole ethyl urea inhibitors of bacterial type II topoisomerases.

The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 µg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 µg/ml for Streptococcus pneumoniae to 0.25 µg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10(-10) with compound A and <5.8 × 10(-11) with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.

Overcoming target-mediated quinolone resistance in topoisomerase IV by introducing metal-ion-independent drug-enzyme interactions.

Quinolones, which target gyrase and topoisomerase IV, are the most widely prescribed antibacterials worldwide. Unfortunately, their use is threatened by the increasing prevalence of target-mediated drug resistance. Greater than 90% of mutations that confer quinolone resistance act by disrupting enzyme-drug interactions coordinated by a critical water-metal ion bridge. Quinazolinediones are quinolone-like drugs but lack the skeletal features necessary to support the bridge interaction. These compounds are of clinical interest, however, because they retain activity against the most common quinolone resistance mutations. We utilized a chemical biology approach to determine how quinazolinediones overcome quinolone resistance in Bacillus anthracis topoisomerase IV. Quinazolinediones that retain activity against quinolone-resistant topoisomerase IV do so primarily by establishing novel interactions through the C7 substituent, rather than the drug skeleton. Because some quinolones are highly active against human topoisomerase IIα, we also determined how clinically relevant quinolones discriminate between the bacterial and human enzymes. Clinically relevant quinolones display poor activity against topoisomerase IIα because the human enzyme cannot support drug interactions mediated by the water-metal ion bridge. However, the inclusion of substituents that allow quinazolinediones to overcome topoisomerase IV-mediated quinolone resistance can cause cross-reactivity against topoisomerase IIα. Therefore, a major challenge in designing drugs that overcome quinolone resistance lies in the ability to identify substituents that mediate strong interactions with the bacterial, but not the human, enzymes. On the basis of our understanding of quinolone-enzyme interactions, we have identified three compounds that display high activity against quinolone-resistant B. anthracis topoisomerase IV but low activity against human topoisomerase IIα.

Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport.

Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first 'open clamp' structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important ß-pinwheel DNA-binding domain. The protein N gate is present in an 'arms-wide-open' state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

Distinct regions of the Escherichia coli ParC C-terminal domain are required for substrate discrimination by topoisomerase IV.

Type IIA DNA topoisomerases are essential enzymes that use ATP to maintain chromosome supercoiling and remove links between sister chromosomes. In Escherichia coli, the type IIA topoisomerase topo IV rapidly removes positive supercoils and catenanes from DNA but is significantly slower when confronted with negatively supercoiled substrates. The ability of topo IV to discriminate between positively and negatively supercoiled DNA requires the C-terminal domain (CTD) of one of its two subunits, ParC. To determine how the ParC CTD might assist with substrate discrimination, we identified potential DNA interacting residues on the surface of the CTD, mutated these residues, and tested their effect on both topo IV enzymatic activity and DNA binding by the isolated domain. Surprisingly, different regions of the ParC CTD do not bind DNA equivalently, nor contribute equally to the action of topo IV on different types of DNA substrates. Moreover, we find that the CTD contains an autorepressive element that inhibits activity on negatively supercoiled and catenated substrates, as well as a distinct region that aids in bending the DNA duplex that tracks through the enzyme's nucleolytic center. Our data demonstrate that the CTD is essential for proper engagement of both gate and transfer segment DNAs, reconciling different models to explain how topo IV discriminates between distinct DNAs topologies.

MIDGET connects COP1-dependent development with endoreduplication in Arabidopsis thaliana.

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

A structurally novel set of inhibitors of bacterial type II topoisomerases with potent in vitro and in vivo antibacterial activity was developed. Dual-targeting ability, hERG inhibition, and pharmacokinetic properties were also assessed.

The role of DNA bending in type IIA topoisomerase function.

Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile172) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.

The MukB-ParC interaction affects the intramolecular, not intermolecular, activities of topoisomerase IV.

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.