RESUMO
Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).
Assuntos
Códon sem Sentido , Doenças Genéticas Inatas , Guanidinas , Quinazolinas , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Gentamicinas/farmacologia , Guanidinas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacologiaRESUMO
The Na(+)/I(-) symporter (NIS) mediates iodide uptake into thyroid follicular cells. Although NIS has been cloned and thoroughly studied at the molecular level, the biochemical processes involved in post-translational regulation of NIS are still unknown. The purpose of this study was to identify and characterize inhibitors of NIS function. These small organic molecules represent a starting point in the identification of pharmacological tools for the characterization of NIS trafficking and activation mechanisms. The screening of a collection of 17,020 druglike compounds revealed new chemical inhibitors with potencies down to 40 nM. Fluorescence measurement of membrane potential indicates that these inhibitors do not act by disrupting the sodium gradient. They allow immediate and total iodide discharge from preloaded cells in accord with a specific modification of NIS activity, probably through distinct mechanisms.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Bibliotecas de Moléculas Pequenas/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Concentração Inibidora 50 , Iodetos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z' greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF(6)(-) > ClO(4)(-) > BF(4)(-) > SCN(-) >> NO(3)(-) > IO(4)(-) > N(3)(-) >> Br(-), in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide symporter.