RESUMO
Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Hidrazonas/metabolismo , Hidrazonas/farmacologia , Nucleotídeos/metabolismo , Fenilalanina , Deleção de Sequência , Animais , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrazonas/química , Hidrazonas/uso terapêutico , Camundongos , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , TemperaturaRESUMO
Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.
Assuntos
Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Ensaios de Triagem em Larga Escala/normas , Modelos Estatísticos , Projetos de Pesquisa , Animais , Sistema Livre de Células/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Reações Falso-Positivas , Imunofluorescência/métodos , Imunofluorescência/normas , Imunofluorescência/estatística & dados numéricos , Ensaios de Triagem em Larga Escala/métodos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/farmacologia , Curva ROC , Distribuição AleatóriaRESUMO
High-throughput small-molecule screens hold great promise for identifying compounds with potential therapeutic value in the treatment of protein-trafficking diseases such as cystic fibrosis (CF) and nephrogenic diabetes insipidus (NDI). The approach usually involves expressing the mutant form of the gene in cells and assaying function in a multiwell format when cells are exposed to libraries of compounds. Although such functional assays are useful, they do not directly test the ability of a compound to correct defective trafficking of the protein. To address this we have developed a novel corrector-screening assay for CF, in which the appearance of the mutant protein at the cell surface is measured. We used this assay to screen a library of 2000 compounds and have isolated several classes of trafficking correctors that had not previously been identified. This novel screening approach to protein-trafficking diseases is robust and general, and could enable the selection of molecules that could be translated rapidly to a clinical setting.