The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a ...The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.展开更多
The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFT...The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane(BCO) G551D-CFTR activators(G551D_ BCOs) with K_d down to 1 μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551D_ BCOs by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551D_ BCOs-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551D_ BCOs; (2) G551D_ BCOs and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.展开更多
基金the Start- up Fund for Returned Overseas Scholars from Northeast Normal U niversity,National ScienceFund for Distinguished Young Scholars(No.30 32 5 0 11) ,Distinguished Young Scholars Fund of Jilin Province(No.2 0 0 30 112 ) ,Excellent Young Teachers Pr
文摘The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.
文摘The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane(BCO) G551D-CFTR activators(G551D_ BCOs) with K_d down to 1 μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551D_ BCOs by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551D_ BCOs-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551D_ BCOs; (2) G551D_ BCOs and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.
