RESUMO
Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes-the bacterial type II topoisomerases gyrase and topoisomerase IV-may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water-metal ion bridge that has been found to mediate the major point of interaction between Escherichia coli topoisomerase IV and Bacillus anthracis topoisomerase IV and gyrase, as well as Mycobacterium tuberculosis gyrase, exists in E. coli gyrase. This is the first investigation of the water-metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water-metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive B. anthracis gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water-metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility.
Assuntos
Quinolonas , Quinolonas/farmacologia , Quinolonas/química , DNA Topoisomerase IV/metabolismo , Escherichia coli/metabolismo , Água/química , Antibacterianos/farmacologia , Antibacterianos/química , Metais/química , Fluoroquinolonas/farmacologia , DNA Girase , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , DNA Topoisomerases Tipo II/metabolismoRESUMO
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.
Assuntos
DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , DNA Topoisomerases Tipo I/química , Fluoroquinolonas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Animais , Antineoplásicos/química , Apoptose , Proliferação de Células , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Feminino , Fluoroquinolonas/química , Humanos , Camundongos , Camundongos Nus , Inibidores da Topoisomerase I/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrA(A90) and GyrA(D94). M. tuberculosis gyrase lacks a conserved serine that anchors a water-metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrA(A90)) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone-gyrase interactions. Clinically relevant mutations at GyrA(A90) and GyrA(D94) cause quinolone resistance by disrupting the bridge-enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone-enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrA(A90V) and GyrA(D94G). 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug-enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , DNA Girase/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Metais/química , Moxifloxacina , Mycobacterium tuberculosis/enzimologia , Água/químicaRESUMO
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 fluoroquinolone-enzyme 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.
Assuntos
Antibacterianos/metabolismo , DNA Girase/metabolismo , Fluoroquinolonas/metabolismo , Mycobacterium tuberculosis/enzimologia , Antibacterianos/química , Cristalografia por Raios X , DNA Girase/química , Fluoroquinolonas/química , Estrutura Molecular , Moxifloxacina , Mycobacterium tuberculosis/metabolismoRESUMO
Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.
Assuntos
DNA Girase/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Fluoroquinolonas/química , Tirosina/química , Sítios de Ligação , Domínio Catalítico , DNA Girase/química , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/síntese química , Fluoroquinolonas/metabolismo , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.
Assuntos
Antibacterianos/química , DNA Girase/genética , Fluoroquinolonas/química , Inibidores da Topoisomerase II/química , Antibacterianos/farmacologia , DNA Girase/química , Dinitrofenóis/química , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fluoroquinolonas/farmacologia , Mutação , Inibidores da Topoisomerase II/farmacologiaRESUMO
Gyrase appears to be the primary cellular target for quinolone antibacterials in multiple pathogenic bacteria, including Bacillus anthracis, the causative agent of anthrax. Given the significance of this type II topoisomerase as a drug target, it is critical to understand how quinolones interact with gyrase and how specific mutations lead to resistance. However, these important issues have yet to be addressed for a canonical gyrase. Therefore, we utilized a mechanistic approach to characterize interactions of quinolones with wild-type B. anthracis gyrase and enzymes containing the most common quinolone resistance mutations. Results indicate that clinically relevant quinolones interact with the enzyme through a water-metal ion bridge in which a noncatalytic divalent metal ion is chelated by the C3/C4 keto acid of the drug. In contrast to other bacterial type II topoisomerases that have been examined, the bridge is anchored to gyrase primarily through a single residue (Ser85). Substitution of groups at the quinolone C7 and C8 positions generated drugs that were less dependent on the water-metal ion bridge and overcame resistance. Thus, by analyzing the interactions of drugs with type II topoisomerases from individual bacteria, it may be possible to identify specific quinolone derivatives that can overcome target-mediated resistance in important pathogenic species.
Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , DNA Topoisomerases Tipo II/química , Farmacorresistência Bacteriana , Quinolonas/química , Inibidores da Topoisomerase II/química , Bacillus anthracis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismoRESUMO
BACKGROUND: Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. METHODS: We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV. RESULTS: In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA. CONCLUSIONS AND GENERAL SIGNIFICANCE: At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding.
Assuntos
Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerase IV/metabolismo , Fluoroquinolonas/farmacologia , Magnésio/metabolismo , Sítios de Ligação , Cálcio/farmacologia , Catálise , Magnésio/farmacologiaRESUMO
The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N-arylacyl O-sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated.
Assuntos
Aminoglicosídeos/metabolismo , Catepsina G/metabolismo , Elastase de Leucócito/metabolismo , Mieloblastina/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Lesão Pulmonar Aguda/metabolismo , Aminoglicosídeos/isolamento & purificação , Catepsina G/antagonistas & inibidores , Fibrose Cística/metabolismo , Humanos , Inflamação/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Mieloblastina/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
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.
Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , DNA Topoisomerases Tipo II/química , Fluoroquinolonas/química , Inibidores da Topoisomerase II/química , Bacillus anthracis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Humanos , Mutação , Inibidores da Topoisomerase II/farmacologiaRESUMO
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-Cys(466) 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-Gly(81) and GyrB-Glu(466) 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.
Assuntos
DNA Girase/metabolismo , DNA Bacteriano/metabolismo , Fluoroquinolonas/metabolismo , Substâncias Macromoleculares/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Ciprofloxacina/farmacologia , Cristalografia por Raios X , DNA Girase/química , DNA Girase/genética , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/química , Fluoroquinolonas/farmacologia , Substâncias Macromoleculares/química , Substâncias Macromoleculares/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mutação , Mycobacterium smegmatis/efeitos dos fármacos , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/metabolismo , Inibidores da Topoisomerase II/farmacologiaRESUMO
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, GrlA(E85K), GrlA(S81F/E85K), GrlA(E85A), GrlA(S81F/E85A) and GrlA(S81F) Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg(2+) 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.
Assuntos
Antibacterianos/química , DNA Topoisomerase IV/química , DNA Topoisomerase IV/efeitos dos fármacos , Metais/química , Quinolonas/química , Antibacterianos/farmacologia , Bacillus anthracis/enzimologia , Cátions Bivalentes/química , Ciprofloxacina/química , Ciprofloxacina/farmacologia , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , Resistência a Medicamentos , Magnésio/química , Mutação , Quinazolinonas/química , Quinazolinonas/farmacologia , Água/químicaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Farmacorresistência Bacteriana , Quinolonas/farmacologia , Animais , Bactérias/enzimologia , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , HumanosRESUMO
Although quinolones have been in clinical use for decades, the mechanism underlying drug activity and resistance has remained elusive. However, recent studies indicate that clinically relevant quinolones interact with Bacillus anthracis (Gram-positive) topoisomerase IV through a critical water-metal ion bridge and that the most common quinolone resistance mutations decrease drug activity by disrupting this bridge. As a first step toward determining whether the water-metal ion bridge is a general mechanism of quinolone-topoisomerase interaction, we characterized drug interactions with wild-type Escherichia coli (Gram-negative) topoisomerase IV and a series of ParC enzymes with mutations (S80L, S80I, S80F, and E84K) in the predicted bridge-anchoring residues. Results strongly suggest that the water-metal ion bridge is essential for quinolone activity against E. coli topoisomerase IV. Although the bridge represents a common and critical mechanism that underlies broad-spectrum quinolone function, it appears to play different roles in B. anthracis and E. coli topoisomerase IV. The water-metal ion bridge is the most important binding contact of clinically relevant quinolones with the Gram-positive enzyme. However, it primarily acts to properly align clinically relevant quinolones with E. coli topoisomerase IV. Finally, even though ciprofloxacin is unable to increase levels of DNA cleavage mediated by several of the Ser80 and Glu84 mutant E. coli enzymes, the drug still retains the ability to inhibit the overall catalytic activity of these topoisomerase IV proteins. Inhibition parallels drug binding, suggesting that the presence of the drug in the active site is sufficient to diminish DNA relaxation rates.
Assuntos
Ciprofloxacina/metabolismo , DNA Topoisomerase IV/metabolismo , Escherichia coli/enzimologia , Metais/química , Água/química , Biocatálise , DNA/químicaRESUMO
The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an â¼ 3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.
Assuntos
Bacillus anthracis/química , DNA Topoisomerase IV/química , Manganês/química , Mutação , Níquel/química , Subunidades Proteicas/química , Água/química , Alanina/química , Alanina/genética , Antibacterianos/química , Bacillus anthracis/enzimologia , Cátions Bivalentes , Ciprofloxacina/química , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/genética , DNA Bacteriano/química , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/química , Modelos Moleculares , Moxifloxacina , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inibidores da Topoisomerase/química , Valina/química , Valina/genéticaRESUMO
We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatias/prevenção & controle , Mitocôndrias Hepáticas/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Ubiquinona/análogos & derivados , Aumento de Peso/efeitos dos fármacos , Animais , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Hepatopatias/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/enzimologia , Compostos Organofosforados/uso terapêutico , Ubiquinona/farmacologia , Ubiquinona/uso terapêutico , Aumento de Peso/fisiologiaRESUMO
BACKGROUND: One way to address the growing problem of antimicrobial resistance is to revive old compounds that may have intrinsic lethal activity that is obscured by protective factors. Bicyclomycin is an old inhibitor of the Rho transcription terminator that by itself shows little rapid lethal activity. However, bicyclomycin participates in bacteriostatic synergy, which raises the possibility that conditions for lethal synergy may exist, perhaps through a suppression of protective factors. METHODS: Bicyclomycin was combined with bacteriostatic inhibitors of gene expression, and bactericidal activity was measured with several cultured Gram-negative pathogens. RESULTS: When used alone, bicyclomycin failed to rapidly kill growing cultures of Escherichia coli; however, the additional presence of bacteriostatic concentrations of tetracycline, chloramphenicol or rifampicin led to rapid killing. Four other pathogen species, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica serotype Typhimurium and Shigella dysenteriae, also exhibited enhanced killing when bicyclomycin was combined with tetracycline or rifampicin. This lethal synergy was achieved at low concentrations (slightly above the MIC) for all agents tested in combinations. Follow-up work with E. coli indicated that lethal synergy arose from a blockage of transcription elongation. Moreover, lethal synergy was reduced when bicyclomycin was added 60 min before tetracycline, suggesting that bicyclomycin induces a protective factor. CONCLUSIONS: The action of bicyclomycin illustrates the potential present in a largely abandoned antibacterial agent; it exhibits lethal synergy when coadministered with known, bacteriostatic inhibitors of gene expression. The identification of protective factors, which are currently uncharacterized, may reveal new ways to promote the lethal action of some old antibiotics.
Assuntos
Antibacterianos/farmacologia , Sinergismo Farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bactérias Gram-Negativas/fisiologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Malaria is a disease that has a major impact in many developing nations, especially on the African continent. There is a need to develop new therapeutics and prophylactic treatments against it. A trisubstituted pyrrole was recently found to inhibit infection of mammalian hepatocytes by Plasmodium sporozoites, but the target of this agent is not known. In this study trisubstituted pyrrole derivatives with different substituents on a piperidinyl nitrogen were prepared. We determined if modifications of the piperidinyl nitrogen would accommodate a drug-biotin linking strategy for affinity purification of the trisubstituted pyrrole's target protein(s).
Assuntos
Plasmodium/metabolismo , Proteínas de Protozoários/química , Pirróis/química , Animais , Hepatócitos/parasitologia , Nitrogênio/química , Piperidinas/química , Plasmodium/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Pirróis/síntese química , Pirróis/farmacologia , Esporozoítos/efeitos dos fármacos , Esporozoítos/metabolismoRESUMO
Insulin resistance (IR) is the root cause of type II diabetes, yet no safe treatment is available to address it. Using a high throughput compatible assay that measures real-time translocation of the glucose transporter glucose transporter 4 (GLUT4), we identified small molecules that potentiate insulin action. In vivo, these insulin sensitizers improve insulin-stimulated GLUT4 translocation, glucose tolerance, and glucose uptake in a model of IR. Using proteomic and CRISPR-based approaches, we identified the targets of those compounds as Unc119 proteins and solved the structure of Unc119 bound to the insulin sensitizer. This study identifies compounds that have the potential to be developed into diabetes treatment and establishes Unc119 proteins as targets for improving insulin sensitivity.