RESUMEN
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Girasa de ADN/química , Quinolinas/química , Quinolinas/farmacología , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II , Antibacterianos/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , División del ADN , Girasa de ADN/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Diseño de Fármacos , Resistencia a Medicamentos , Escherichia coli/enzimología , Manganeso/metabolismo , Modelos Moleculares , Conformación Proteica , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Relación Estructura-ActividadRESUMEN
Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.
Asunto(s)
Antimaláricos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Plasmodium/enzimología , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Animales , Antimaláricos/química , Clonación Molecular , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Cinética , Estructura Molecular , Plasmodium/clasificación , Relación Estructura-Actividad , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
Asunto(s)
Antígenos de Neoplasias/aislamiento & purificación , Clonación Molecular/métodos , ADN-Topoisomerasas de Tipo II/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Adenosina Difosfato/metabolismo , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Baculoviridae/genética , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , ADN Circular/química , ADN Circular/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Concentración 50 Inhibidora , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Espectrometría de Fluorescencia , Spodoptera/metabolismoRESUMEN
Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.
Asunto(s)
Bioensayo/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Western Blotting , Línea Celular , Humanos , Quinasa I-kappa B/metabolismo , Modelos Biológicos , FN-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Fosforilación , Spodoptera , Quinasa de Factor Nuclear kappa BRESUMEN
Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15â Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.
Asunto(s)
División del ADN , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos de Espiro/química , Staphylococcus aureus/enzimología , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , MoxifloxacinoRESUMEN
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II/química , Antibacterianos/farmacología , Antineoplásicos/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana , Etopósido/farmacología , Fluoroquinolonas/farmacología , Modelos Moleculares , Estructura Molecular , Moxifloxacino , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.
Asunto(s)
Antibacterianos/síntesis química , Enterococcus/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Metionina-ARNt Ligasa/antagonistas & inhibidores , Quinolonas/síntesis química , Staphylococcus/efectos de los fármacos , Absceso/tratamiento farmacológico , Absceso/microbiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Quinolonas/química , Quinolonas/farmacología , Ratas , Ratas Sprague-Dawley , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Relación Estructura-ActividadRESUMEN
Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Espectrometría de Masas/métodos , Ciclooxigenasa 2/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Colorantes Fluorescentes/metabolismo , Humanos , Concentración 50 Inhibidora , Oxidorreductasas Intramoleculares/metabolismo , Prostaglandina-E SintasasRESUMEN
High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified ethanolamine 1 as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.
Asunto(s)
Antibacterianos/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Etanolaminas/síntesis química , Etanolaminas/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Staphylococcus aureus/enzimología , Animales , Antibacterianos/farmacología , Diseño de Fármacos , Cinética , Mamíferos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Sensibilidad y Especificidad , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The design of conformationally restricted eight-membered ring diketones as transition state mimics of the mechanism of action of cyclotheonamides on serine proteases is described. Two target compounds are prepared from mutilin, derived from the natural product pleuromutilin. Compound 3 shows significant inhibition of plasmin and urokinase in enzyme rate assays, but an analogue 4 in which the amide moiety has been omitted does not. An X-ray crystal structure of the diketone 3 confirms the conformational predictions made by molecular modelling.
Asunto(s)
Cetonas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Hidrocarburos Aromáticos con Puentes/síntesis química , Hidrocarburos Aromáticos con Puentes/farmacología , Diseño de Fármacos , Cetonas/química , Cetonas/farmacología , Cinética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Compuestos Policíclicos/química , Compuestos Policíclicos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.