F508-Cftr Modulator Screen Based On Cell Surface Targeting Of A Chimeric Nucleotide Binding Domain 1 Reporter

The most common cystic fibrosis-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (Delta F508). The Delta F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify Delta F508-CFTR modulators that act on NBD1. A biochemical screen for Delta F508-NBD1 cell surface expression was done in Madin-Darby canine kidney cells expressing a chimeric reporter consisting of Delta F508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z' factor of 0.7. The screening of similar to 20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased Delta F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length Delta F508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify Delta F508-CFTR modulators that target the NBD1 domain.