Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields ΔF508-CFTR Correctors That Augment VX-809 Maximal Efficacy

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, Delta F508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membranespanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2difluoro- 1,3-benzodioxol-5-yl) cyclopropyl] carbonyl] amino]-3methyl- 2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation Delta F508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for Delta F508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface Delta F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 mu M. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in.F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a Delta F508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the therapeutic ceiling of first-generation correctors.