Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone–enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.
2The fluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial resistance erodes the effectiveness of other agents (fluoroquinolone sales accounted for 18% of the antibacterial market in 2006) (41). One of the attractive features of the quinolones is their ability to kill bacteria rapidly, an ability that differs widely among the various derivatives. For example, quinolones differ in rate and extent of killing, in the need for aerobic metabolism to kill cells, and in the effect of protein synthesis inhibitors on quinolone lethality. Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives.Before describing the types of damage caused by the quinolones, it is useful to define lethal activity. Operationally, it is the ability of drug treatment to reduce the number of viable cells, usually measured as CFU on drug-free agar after treatment. This assay is distinct from measurements that detect inhibition of growth (e.g., MIC), since with the latter bacteria are exposed to drug throughout the measurement. The distinction between killing and blocking growth is important because it allows susceptibility determinations to be related to particular biological processes. For example, inhibition of growth is typically reversed by the removal of drug, while cell death is not. Thus, biochemical events associated with blocking growth should be readily reversible, while those responsible for cell death should be difficult to reverse. Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure. Moreover, protective functions, such as repair and stress responses, can be distinguished by whether their absence affects inhibition of growth, killing, or both.The intracellular targets of the quinolones are two DNA topoisomerases: gyrase and topoisomerase IV. Gyrase tends to be the primary target in gram-negative bacteria, while topoisomerase IV is preferentially inhibited by most quinolones in gram-positive organisms (28). Both enzymes use a doublestrand DNA passage mechanism, and it is likely that quinolone biochemistry is similar for both. However, physiological differences between the enzymes exist, some of which may bear on quinolone lethality.In the present minireview we consider cell death through a two-part "poison" hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage. Other consequences of quinolone treatment, such as depletion of gyrase and topoisomerase IV activity, are probably less immediate (42). To provide a framework for considering quinolone lethality, we begin by briefly describing the drug-topoisomerase-DNA complexes. Readers interested in a more comprehensive discussion of quinolones are referred to a previously published work (28). QUINOLONE-TOPOISOMERASE-DNA COMPLEXESAs a normal part of their reaction mechanism, g...
The quinolones trap DNA gyrase and DNA topoisomerase IV on DNA as complexes in which the DNA is broken but constrained by protein. Early studies suggested that drug binding occurs largely along helix-4 of the GyrA (gyrase) and ParC (topoisomerase IV) proteins. However, recent X-ray crystallography shows drug intercalating between the -1 and +1 nucleotides of cut DNA, with only one end of the drug extending to helix-4. These two models may reflect distinct structural steps in complex formation. A consequence of drug-enzyme-DNA complex formation is reversible inhibition of DNA replication; cell death arises from subsequent events in which bacterial chromosomes are fragmented through two poorly understood pathways. In one pathway, chromosome fragmentation stimulates excessive accumulation of highly toxic reactive oxygen species that are responsible for cell death. Quinolone resistance arises stepwise through selective amplification of mutants when drug concentrations are above the MIC and below the MPC, as observed with static agar plate assays, dynamic in vitro systems, and experimental infection of rabbits. The gap between MIC and MPC can be narrowed by compound design that should restrict the emergence of resistance. Resistance is likely to become increasingly important, since three types of plasmid-borne resistance have been reported.
Vancomycin is an important drug for the treatment of Gram-positive bacterial infections. Resistance to vancomycin has begun to appear, posing a serious public health threat. Vancomycin analogs containing modified carbohydrates are very active against resistant microorganisms. Results presented here show that these carbohydrate derivatives operate by a different mechanism than vancomycin; moreover, peptide binding is not required for activity. It is proposed that carbohydrate-modified vancomycin compounds are effective against resistant bacteria because they interact directly with bacterial proteins involved in the transglycosylation step of cell wall biosynthesis. These results suggest new strategies for designing glycopeptide antibiotics that overcome bacterial resistance.
Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlAS81F and GrlAS81Y quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg2+ bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg2+ concentrations to support DNA cleavage by GrlAS81F topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the ternary enzyme-quinolone-DNA complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg2+ bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically-relevant quinolone resistance mutations in bacterial type II topoisomerases.
Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlAE85K, GrlAS81F/E85K, GrlAE85A, GrlAS81F/E85A and GrlAS81F Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg2+ concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein–quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.
Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and in 2013 accounted for 1.5 million deaths. Fluoroquinolone antibacterials, which target DNA gyrase, are critical agents used to halt the progression from multidrug-resistant tuberculosis to extensively resistant disease; however, fluoroquinolone resistance is emerging and new ways to bypass resistance are required. To better explain known differences in fluoroquinolone action, the crystal structures of the WT Mtb DNA gyrase cleavage core and a fluoroquinolone-sensitized mutant were determined in complex with DNA and five fluoroquinolones. The structures, ranging from 2.4-to 2.6-Å resolution, show that the intrinsically low susceptibility of Mtb to fluoroquinolones correlates with a reduction in contacts to the water shell of an associated magnesium ion, which bridges fluoroquinolone-gyrase interactions. Surprisingly, the structural data revealed few differences in fluoroquinoloneenzyme contacts from drugs that have very different activities against Mtb. By contrast, a stability assay using purified components showed a clear relationship between ternary complex reversibility and inhibitory activities reported with cultured cells. Collectively, our data indicate that the stability of fluoroquinolone/DNA interactions is a major determinant of fluoroquinolone activity and that moieties that have been appended to the C7 position of different quinolone scaffolds do not take advantage of specific contacts that might be made with the enzyme. These concepts point to new approaches for developing quinolone-class compounds that have increased potency against Mtb and the ability to overcome resistance.Mycobacterium tuberculosis | antibiotic resistance | fluoroquinolone | gyrase | complex stability
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. Based on 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.
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