Drugs in the gland secretion experiments were diluted with bath solution immediately before use at the concentrations indicated

This study has revealed the IP inhibitor family as a promising anti-tubercular agent, targeting an essential component of the electron transport chain, QcrB. Due to the nature of the target, it is conceivable that the IP compounds could target both active and latent phases of TB infection, an important requirement of future anti-tubercular agents. In most cultured cell models and native epithelia, a small portion of the F508del protein can escape the quality control system of the ER, and subsequently undergo complex glycosylation in the Golgi compartment and transfer to the apical membrane of epithelial cells. The F508del protein at the cell surface is active as a chloride channel, though with a strongly reduced open probability and considerably higher turnover rate as compared to wild type CFTR. The instability of rescued F508del CFTR was attributed to unfolding and subsequent ubiquitination, endocytosis, and lysosomal degradation by a peripheral protein QC system sharing multiple chaperones and co-chaperones with the QC in the ER. Attempts to correct the F508del CFTR allelespecific phenotype are currently focussed on the selection of compounds that overcome the inefficient folding of the mutant protein, or enhance the CFTR chloride channel activity. Small molecule correctors may also act as pharmacological chaperones and enhance the cellsurface stability of F508del-CFTR. Partial rescue of the human F508del CFTR protein has been demonstrated in cell culture using different strategies. Initially, restoration of F508del CFTR processing was accomplished by low temperature incubation. Subsequently, competition with truncated CFTR constructs, chemical chaperones, transcriptional regulators, pharmacological chaperones or modification of available lead compounds. Most of these studies have been performed with primary or immortalised human airway cells in vitro. Recent studies showed that the relative efficacy of different types of correctors depends on the cell type and experimental context. This limits the predictive value of in vitro data for clinical applications assays and stresses the importance of choosing models that reflect the in vivo situation. Prior to expensive and potentially harmful clinical trials, testing of a promising candidate in the context of a fully differentiated organ is advisable. In this study we have used the F508del mouse strain generated at the Erasmus MC Rotterdam for this purpose. In this model, made by ‘hit and run’ homologous recombination, a F508del mutant form of Cftr mRNA is present at normal expression levels in all affected organs. To avoid complications related to unfavourable pharmacokinetics of candidate correctors or potentiators that may obscure effects on mutant CFTR activity during in vivo application, a major effort in our laboratory has been to define assay conditions allowing the study of F508del CFTR processing and rescue in murine intestinal epithelium in maintenance culture. The assay we have developed can be used to study the trafficking defect of the F508del protein in intact epithelial tissue. Here we show for the first time that low temperature incubation induced the delivery of complex-glycosylated F508del CFTR to the plasma membrane and full functional rescue of CFTR activity in mouse intestinal mucosa. Furthermore, we show that at physiological temperature this rescue action could be mimicked by a variety of proteasome inhibitors. The latter BYL719 PI3K inhibitor result contrasts with earlier reports using cell culture models, but confirms a later study in a different immortalized cell population. This emphasizes the importance of our approach to examine F508del CFTR rescue in fully differentiated tissue. This preclinical study clearly demonstrates that interfering with the proteolytic activity of the proteasome may restore CFTR function in intact murine epithelium, and suggests that proteasome inhibitors may have therapeutic potential for the treatment of CF. Initially, pulse-chase experiments of CFTR transfected HEK and CHO cells showed the accumulation of detergent-insoluble, multi-ubiquitinylated CFTR molecules with no detectable increase of folded CFTR after proteasome inhibition. However, more recent studies by Gentzsch et al. on the endocytic trafficking of wild type and F508del CFTR in BHK cells demonstrated that proteasome inhibitors can stabilize low temperature rescued mutant protein by preventing lysosomal degradation and promoting recycling of endocytosed CFTR back to the plasma membrane. Comparable results were presented by Vij et al in a similar immortalized cell line.

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