ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

Abstract

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (ΔF508). The ΔF508 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 ΔF508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-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 ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ΔF508-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 ΔF508-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 ΔF508-CFTR modulators that target the NBD1 domain.

One Size Does Not Fit All: The Past, Present and Future of Cystic Fibrosis Causal Therapies

“…F508del—and a number of other processing mutations—however, are known to also suffer from gating defects [ 54 ]. To account for this, screening assays looking directly at the trafficking of mutant CFTR proteins have since also been developed [ 128 , 129 , 130 , 131 , 132 ]. Recently, a fluorescence resonance energy transfer (FRET) based HTS platform was developed to evaluate the folding of nascent, ribosome-bound NBD1 mutants to specifically identify compounds able to restore the conformation of NBD1 [ 133 ].…”

Overview of CFTR activators and their recent studies for dry eye disease: a review

“…Substantiating their potential, all four compounds were validated as CFTR potentiators through short-circuit current recordings performed within a comprehensive Phe508del-CFTR potentiator assay. 43…”

“…Substantiating their potential, all four compounds were validated as CFTR potentiators through short-circuit current recordings performed within a comprehensive Phe508del-CFTR potentiator assay. 43 Yang et al first reported the CFTR-activating activity of 1,4-dihydropyridine structural compounds through HTS. 40 Pedemonte et al identified several antihypertensive 1,4-dihydropyridines (DHPs) such as nifedipine, nicardipine, nimodipine, isradipine, nitrendipine, felodipine, and niguldipine as potential ΔF508-CFTR activators (Fig.…”

Current development of CFTR potentiators in the last decade

“…Thiophenes moiety emerged again in a study that was specifically focused on the NBD1 domain of CFTR. [51] Instead of using the whole CFTR protein, the authors designed a special construct consisting of NBD1, carrying the Phe508del mutation, fused to CD4 transmembrane domain and an extracellular horseradish peroxidase (HRP) reporter. Using luminescence, a library of 20,000 compounds was screened to find novel correctors.…”