Rescue of functional CFTR channels in cystic fibrosis: a dramatic multivalent effect using iminosugar cluster-based correctors.

Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N-butyl 1-deoxynojirimycin (N-Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized. Significantly, the trivalent analogues were found to be up to 225-fold more potent than N-Bu DNJ and up to 1000-fold more potent than the corresponding monovalent models. These results provide the first description of a multivalent effect for correcting protein folding defects in cells and should have application for the treatment of a number of protein folding disorders. Preliminary mechanistic studies indicated that CFTR correction activity enhancement was not due to a multivalent effect in ER-glucosidase inhibition or to a different mode of action of the multivalent iminosugars.

Transient receptor potential canonical channel 6 links Ca2+ mishandling to cystic fibrosis transmembrane conductance regulator channel dysfunction in cystic fibrosis.

In cystic fibrosis (CF), abnormal control of cellular Ca(2+) homeostasis is observed. We hypothesized that transient receptor potential canonical (TRPC) channels could be a link between the abnormal Ca(2+) concentrations in CF cells and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We measured the TRPC and CFTR activities (using patch clamp and fluorescent probes) and interactions (using Western blotting and co-immunoprecipitation) in CF and non-CF human epithelial cells treated with specific and scrambled small interfering RNA (siRNA). The TRPC6-mediated Ca(2+) influx was abnormally increased in CF compared with non-CF cells. After correction of abnormal F508 deletion (del)-CFTR trafficking in CF cells, the level of TRPC6-dependent Ca(2+) influx was also normalized. In CF cells, siRNA-TRPC6 reduced this abnormal Ca(2+) influx. In non-CF cells, siRNA-TRPC6 reduced the Ca(2+) influx and activity wild-type (wt)-CFTR. Co-immunoprecipitation experiments revealed TRPC6/CFTR and TRPC6/F508 del-CFTR interactions in CF or non-CF epithelial cells. Although siRNA-CFTR reduced the activity of wt-CFTR in non-CF cells and of F508 del-CFTR in corrected CF cells, it also enhanced TRPC6-dependent Ca(2+) influx in non-CF cells, mimicking the results obtained in CF cells. Finally, this functional and reciprocal coupling between CFTR and TRPC6 was also detected in non-CF ciliated human epithelial cells freshly isolated from lung samples. These data indicate that TRPC6 and CFTR are functionally and reciprocally coupled within a molecular complex in airway epithelial human cells. Because this functional coupling is lost in CF cells, the TRPC6-dependent Ca(2+) influx is abnormal.

A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+. In this study, using assays to measure chloride channel activity, we show that guanabenz is an activator of CaCC in freshly dissociated human bronchial epithelial cells from three CF patients with various genotypes (F508del/F508del, F508del/R1066C, F508del/H1085R). We further characterised the effect of guanabenz and show that it is independent of α-adrenergic receptors, is inhibited by the TRPC family inhibitor SKF-96365 but not by the TRPV family inhibitor ruthenium red. Using western-blotting, Ca2+ measurements and iodide efflux assay, we found that TRPC1 siRNA has no effect on guanabenz induced responses whereas TRPC6 siRNA prevented the guanabenz-dependent Ca2+ influx and the CaCC-dependent activity stimulated by guanabenz. In conclusion, we show that TRPC6 channel is pivotal for the activation of CaCC by guanabenz through a α2-adrenergic-independent pathway in human airway epithelial cells. We suggest propose a functional coupling between TRPC6 and CaCC and guanabenz as a potential TRPC6 activator for exploring TRPC6 and CaCC channel functions and corresponding channelopathies.

A new 9-alkyladenine-cyclic methylglyoxal diadduct activates wt- and F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the main chloride channel present in the apical membrane of epithelial cells and the F508 deletion (F508del-CFTR) in the CF gene is the most common cystic fibrosis-causing mutation. In the search for a pharmacotherapy of cystic fibrosis caused by the F508del-CFTR, a bi-therapy could be developed associating a corrector of F508del-CFTR trafficking and an activator of the channel activity of CFTR. Here, we report on the synthesis of 9-alkyladenine derivatives analogues of our previously discovered activator of wt-CFTR and F508del-CFTR, GPact-11a, and the identification of a new activator of these channels, GPact-26a, through various flux assays on human airway epithelial CF and non-CF cell lines and in vivo measurement of rat salivary secretion. This study reveals that the possible modifications of the side chain introduced at the N9 position of the main pharmacophore are highly limited since only an allyl group can replace the propyl side chain present in GPact-11a to lead to a strong activation of wt-CFTR in CHO cells. Docking simulations of the synthesised compounds and of four described modulators performed using a 3D model of the wt-type CFTR protein suggest five possible binding sites located at the interface of the nucleotide binding domains NBD1/NBD2. However, the docking study did not allow the differentiation between active and non-active compounds.