RESUMO
This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Síntese de Ácido Nucleico/isolamento & purificação , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Sistema Livre de Células/metabolismo , Células Cultivadas , Técnicas de Laboratório Clínico , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Inibidores da Síntese de Ácido Nucleico/análise , Fator de Iniciação 2 em Procariotos/antagonistas & inibidores , Fator de Iniciação 2 em Procariotos/fisiologia , Proteínas de Ligação ao Cap de RNA/fisiologia , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Aminoacilação de RNA de Transferência/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismoRESUMO
Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNA(f)(Met) to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNA(f)(Met) blocked alpha-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNA(f)(Met). Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNA(f)(Met) by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNA(f)(Met) and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.
