Optimization of a Yellow Fluorescent Protein-Based Iodide Influx High-Throughput Screening Assay for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators

Abstract

Cystic fibrosis is an inherited, life-threatening disease associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, F508del CFTR, is found in 90% of CF patients. The loss of a single amino acid (phenylalanine at position 508) results in malformed CFTR with defective trafficking to the plasma membrane and impaired channel function. A functional assay with cells expressing F508del CFTR has been previously described by others using genetically engineered halide-sensitive yellow fluorescent protein to screen for CFTR modulators. We adapted this yellow fluorescent protein assay to 384-well plate format with a high-throughput screening plate reader, and optimized the assay in terms of data quality, resolution, and throughput, with target-specific protocols. The optimized assay was validated with reference compounds from cystic fibrosis foundation therapeutics. On the basis of the Z-factor range (≥0.5) and the potential productivity, this assay is well suited for high-throughput screening. It was successfully used to screen for active single agent and synergistic combinations of single agent modulators of F508del CFTR from a library collection of current active pharmaceutical ingredients (supported by Cystic Fibrosis Foundation Therapeutics).

Identification of Synergistic Combinations of F508del Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators

“…The ''corrector'' protocol was designed to detect compounds that enhance CFTR expression and trafficking to the membrane; the ''potentiator'' protocol was designed for detecting agents that increase CFTR channel activities within a short period of treatment (Sui et al, 2010). A ''no-wash'' flux assay protocol was designed to catch indiscriminately the effects of both correctors and potentiators in a single experiment.…”

“…The library included a set of reference compounds with previously demonstrated activity on CFTR (Sui et al, 2010). We adapted the FRT YFP halide flux assay, first described by Galietta 18 and subsequently used by multiple groups to identify CFTR modulators, [43][44][45][46][47] for increased throughput and compatibility with our combination HTS (cHTSÔ) platform (Sui et al, 2010).…”

“…The corrector protocol includes a 24-h incubation period to allow time for compounds to affect F508del CFTR processing and trafficking to the plasma membrane (Sui et al, 2010). A subset of compounds with NQR-fold >1.3 are listed in Table 1.…”

“…We used an improved version of the YFP flux assay 56 (Sui et al, 2010) on an HTS platform to identify correctors and potentiators of F508del CFTR from a library of approved drugs and tool compounds.…”

“…In this study, we sought to identify single compounds and twoway synergistic combinations that modulate F508del CFTR activity, using a modified CFTR yellow fluorescent protein (YFP) flux assay (Sui et al, 2010) 31 in Fischer rat thyroid (FRT) cells. To maximize the potential for rapid clinical translation, we focused on a library of *3,000 approved drugs and tool compounds, many with known safety profile and/or mechanism of action.…”