Amy Schaberg

Potential of denufosol as an early intervention in CF lung disease: efficacy in patients with minimal pharmacotherapy in a US phase 3 clinical trial

77 Potential of denufosol as an early intervention in CF lung disease: efficacy in patients with minimal pharmacotherapy in a US phase 3 clinical trial F.J. Accurso1, W. Tian2, A. Schaberg2, T. Navratil2, M.S. Howenstine3, T.G. Liou4. 1University of Colorado, Denver, CO, United States; 2Inspire, Durham, NC, United States; 3James Whitcomb Riley Hospital for Children, Indianapolis, IN, United States; 4University of Utah, Salt Lake City, UT, United States

Effects of denufosol on sinusitis-related complaints in a phase 3 trial in cystic fibrosis patients

The most common mutation in Cystic Fibrosis, F508del, causes defects in trafficking, channel gating and endocytosis of the CFTR (CF Transmembrane conductance Regulator) protein. We have previously developed an automatic assay to identify agents, termed correctors, directed at repairing these defects. This study shows the identification from our screening of a novel corrector, roscovitine, able to restore the membrane localization and functionality of F508del-CFTR protein on the human airway epithelial CF cell line JME/CF15. We found that roscovitine corrected F508del-CFTR with an EC50 of 56mM. Moreover, biochemical analysis and immunofluorescence imaging confirmed the restoration of a mature F508del-CFTR at the cell surface. To pinpoint the molecular mechanism of roscovitine, we realized competition studies between roscovitine and inhibitors of the endoplasmic reticulum quality control (ERQC). No potentiation of roscovitine correction occured after co-treatment by roscovitine and the degradation inhibitor MG132. Only a small potentiation was obtained after co-treatment by roscovitine and thapsigargin which inhibits interaction between CFTR and calnexin, a Ca2+-dependent ER chaperone. Additional studies showed that roscovitine stimulates an intracellular Ca2+ increase, consecutive of the ER emptying. Moreover a Ca2+-independent inhibition of proteasome activity (~50%) in CF15 cells was observed after a roscovitine treatment. We conclude from the present study that roscovitine restores the abnormal trafficking of F508del-CFTR via a Ca2+-dependent and a Ca2+-independent mechanisms of action allowing disturbance of the ability of the ERQC to interact and to degrade F508del-CFTR proteins. Supported by Vaincre la Mucovicidose and CNRS.