RESUMEN
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
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 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.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Staphylococcus aureus/enzimología , Triptófano-ARNt Ligasa/antagonistas & inhibidores , Antibacterianos/síntesis química , Bacterias/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Hidrólisis , Indicadores y Reactivos , Indoles/síntesis química , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
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.