Improved fluorescence assays to measure the defects associated with F508del‐CFTR allow identification of new active compounds

• Published in: British journal of pharmacology Vol. 174; no. 7; pp. 525 - 539
• Main Authors: Langron, Emily, Simone, Michela I, Delalande, Clémence M S, Reymond, Jean‐Louis, Selwood, David L, Vergani, Paola
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-04-2017; John Wiley and Sons Inc
• Subjects: Aminophenols - analysis; Aminophenols - chemical synthesis; Aminophenols - chemistry; Aminophenols - pharmacology; Bacterial Proteins - analysis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biosynthesis; Cystic fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Drug Evaluation, Preclinical - methods; Fluorescence; Genes; HEK293 Cells; Humans; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Molecular Structure; Mutation; Quinolones - analysis; Quinolones - chemical synthesis; Quinolones - chemistry; Quinolones - pharmacology; Research Paper; Research Papers; Small Molecule Libraries - analysis; Small Molecule Libraries - chemical synthesis; Small Molecule Libraries - chemistry; Small Molecule Libraries - pharmacology
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/bph.13715

Background and Purpose
Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Clˉ/HCO3ˉ channel. F508del, the most common CF‐associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen.
Experimental Approach
HEK293 cells expressing YFP‐F508del‐CFTR, in which halide sensitive YFP is tagged to the N‐terminal of CFTR, were used to screen a small library of compounds based on the VX‐770 scaffold. Cells expressing F508del‐CFTR‐pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators.
Key Results
Active compounds with efficacy ~50% of VX‐770, micromolar potency, and structurally distinct from VX‐770 were identified in the screen. The F508del‐CFTR‐pHTomato assay suggests that the hit compound MS131A, unlike VX‐770, does not decrease membrane exposure of F508del‐CFTR.
Conclusions and Implications
Most known potentiators have a negative influence on F508del‐CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del‐CFTR, and other CF‐causing mutations.