Abstract
The most prevalent CFTR mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain 1 (NBD1) and stability of the interface between NBD1 and the membrane spanning domains (MSDs). The interfacial stability defect can be partially corrected by the investigational drug VX-809 or the R1070W mutation. 'Second-generation' ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing CF 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 ΔF508-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 either in the presence or absence of R1070W. Using a luminescence read-out of HRP activity, screening of ~110,000 small molecules produced 9 novel corrector scaffolds that increased cell surface ΔF508-CFTR expression by up to 200 % in the presence vs. absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with EC50 <5 μM. 8 chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ΔF508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 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.
- The American Society for Pharmacology and Experimental Therapeutics