Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.     

Streptococcus pneumononiae gyrase ATPase: development and validation of an assay for inhibitor discovery and characterization

The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors.     

Site-specific DNA cleavage by Chlorella virus topoisomerase II

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.     

Teratogenicity of antibacterial agents

The aim of our study was to study the possible correlation between use of antibacterial drugs in pregnancy and occurrence of congenital malformations. Among 6099 investigated pregnant women, 392 (6.43%) used antibacterial drugs. The most frequently used antibacterials belonged to category B (75.77%), while 14.54% antibiotics belonged to category D and 1.02% to category X. The most often used antibiotics were cephalexin (22.19%), amoxicillin (20.66%) and ampicillin (14.29%). In 14 embryos exposed to effects of beta-lactams in utero, malformations were detected. The results of this study show possible teratogenic potential even with those antibacterials which are considered safe, but as those are usually minor malformations, they often pass undetected. Because of that and because of frequent use of antibacterials during pregnancy, detailed examinations concerning their safety should be made.     

A scintillation proximity assay amenable for screening and characterization of DNA gyrase B subunit inhibitors.

DNA gyrase is the target of coumarin and cyclothialidine antibacterials, which bind to the B subunit of the enzyme (GyrB). Currently available GyrB inhibitors have not been clinically successful, but their high in vitro potency against DNA gyrase has raised interest in the development of novel noncoumarin antibacterials acting at the same site. We report the development of a simple scintillation proximity assay (SPA) for the study of binding interactions between coumarin or noncoumarin antibacterials and GyrB, which prevents the needs of separation steps and can be run in microtiter plate formats. The assay is based on the detection of the binding of a radioligand, [3H]dihydronovobiocin, to a biotin-labeled 43-kDa fragment of GyrB (biotin-GyrB43), which is captured by streptavidin-coated SPA beads. The typical assay was conducted in 96-well microtiter plates, with final concentration of 10 nM for biotin-GyrB43, 20 nM for [3H]dihydronovobiocin, and 33 microg of SPA beads/well. From saturation experiments, an equilibrium dissociation constant (K(d)) for dihydronovobiocin of 8.10 nM was found. Displacement studies gave 50% inhibitory concentrations (IC(50)) of 42, 64, and 11 nM for novobiocin, dihydronovobiocin, and the cyclothialidine analogue GR122222X, respectively, consistent with previous findings. The assay was found to be robust to dimethyl sulfoxide up to 5% (v/v) and can be used for high-throughput screens of large chemical collections in the search of novel DNA gyrase inhibitors.     

Distinct mechanisms of guanine-specific DNA photodamage induced by nalidixic acid and fluoroquinolone antibacterials

Fluoroquinolone antibacterials, which have been used for the treatment of a variety of infectious diseases, are reported to be photocarcinogenic. We investigated the mechanisms of DNA damage by UVA radiation (365 nm) plus fluoroquinolone antibacterials using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Photocarcinogenic nalidixic acid (NA), which is an old member of synthetic quinolone antibacterials, caused DNA damage specifically at 5'-GG-3' sequences, whereas lomefloxacin (LFLX) did not exhibit the site preference for consecutive guanines. LFLX-induced DNA photodamage was inhibited by sodium azide and enhanced in D2O, suggesting that singlet oxygen plays the key role in the DNA damage. LFLX plus UVA induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) depending on LFLX concentrations, and 8-oxodG formation was enhanced in single-stranded DNA. In contrast, NA induced larger amounts of 8-oxodG in double-stranded DNA. ESR spin destruction method revealed that NA induced DNA photodamage through electron transfer but LFLX did not. These findings indicate that DNA damage induced by photoactivated LFLX and NA plays an important role in expression of their photocarcinogenicity.     

Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193

Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.     

ATP synthase in mycobacteria: Special features and implications for a function as drug target

ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

DNA topoisomerase I from human placenta

In this study we report that human placenta is an excellent source of DNA topoisomerase I. The enzyme can be isolated in the fully intact 100 kDa form although lower molecular mass species are also observed. Occasionally, the enzyme can be resolved into two peaks of activity by chromatography on phosphocellulose. As expected, the enzyme promotes marked cleavage of DNA in response to the anticancer drug camptothecin. Because of this property and the ready availability of human placenta, the enzyme should prove to be useful in the development and testing of new anticancer drugs that target topoisomerase I.     

Sulfa and trimethoprim-like drugs – antimetabolites acting as carbonic anhydrase,dihydropteroate synthase and dihydrofolate reductase inhibitors

Abstract

Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families.     

Squaring up to DNA: pentapeptide repeat proteins and DNA mimicry

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed β-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase—a potentially useful therapeutic property—and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.     

Novel inhibitors of bacterial cell wall synthesis

Over the past forty years, efforts to discover antibacterials have yielded a wide variety of chemical structures, almost exclusively natural products, which inhibit many steps in cell wall synthesis. Although screening for new cell wall inhibitors has been continuous during that period, there have been few reports of new drugs. With the advent of genomics, high resolution X-ray crystallography and the recognition of the need for new antibiotics to combat resistant organisms, there has been a resurgence in interest in this validated target area.     

Clostridium difficile DNA polymerase IIIC: basis for activity of antibacterial compounds

Based on the finding that aerobic Gram-positive antibacterials that inhibit DNA polymerase IIIC (pol IIIC) were potent inhibitors of the growth of anaerobic Clostridium difficile (CD) strains, we chose to clone and express the gene for pol IIIC from this organism. The properties of the recombinant enzyme are similar to those of related pol IIICs from Gram-positive aerobes, e.g. B. subtilis. Inhibitors of the CD enzyme also inhibited B. subtilis pol IIIC, and were competitive with respect to the cognate substrate 2'-deoxyguanosine 5'-triphosphate (dGTP). Significantly, several of these inhibitors of the CD pol IIIC had potent activity against the growth of CD clinical isolates in culture.     

The antibiotic R&D pipeline: an update

There is an urgent need for new antibacterials to target emerging multidrug-resistant bacteria. The need for such agents is rising while the efforts in antibacterial research have declined dramatically in the past few decades with the result of only four compounds belonging to new chemical classes being approved for clinical use. The main reasons that led to this critical situation are shortly described. A renewed interest in the research of new effective antimicrobials is nonetheless delivering compounds deriving mainly from modification of existing drugs, yet new chemical classes are appearing. Because many of these activities have started relatively recently, we should expect a long period before new antibiotics are added to the medical armamentarium.     

The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing

Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.     

Toward Discovering New Anti-Cancer Agents Targeting Topoisomerase IIα: A Facile Screening Strategy Adaptable to High Throughput Platform

Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO₄, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α–mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.     

Analysis of the effects of antibiotics on the structure of nucleosome core particles determined by DNAase I cleavage

Patterns of sensitivity to DNAase I cleavage have been analysed in order to investigate the effects of anti-tumour antibiotics and related drugs on nucleosome core particles containing different DNA restriction fragments. In this article, we review the experimental results which show that after controlled digestion of defined-sequence core particles, new cleavage products appear in the enzyme digestion patterns which lie approximately mid-way between the strong bands characteristic of native nucleosome core particles. The effects of the antibiotics, which include bis-intercalators as well as minor groove-binding ligands (but not monofunctional intercalators), are explained in terms of an induced change in rotational setting (phasing) of the core DNA. The new rotational positioning of DNA induced by antibiotic binding appears to be almost independent of DNA sequence, although some differences can be observed with the various pieces of DNA, most likely reflecting the exact number and disposition of antibiotic binding sites.     

A mutation in human topoisomerase II alpha whose expression is lethal in DNA repair-deficient yeast cells

Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II alpha, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 --> Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 --> Asn Top2alpha has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2alpha(Asp-48 --> Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.     

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).