Optimization of a Yellow fluorescent protein-based iodide influx high-throughput screening assay for cystic fibrosis transmembrane conductance regulator (CFTR) modulators.
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).
Zalicus, Inc., Cambridge, Massachusetts, USA. jinlsui@cfrx.org
Jinliang
J
Sui
Shakira
S
Cotard
Jennifer
J
Andersen
Ping
P
Zhu
Jane
J
Staunton
Margaret
M
Lee
Stephen
S
Lin
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000188:drug therapy* / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D007454:Iodides / Q000378:metabolism*; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000378:metabolism*; D009154:Mutation; D021381:Protein Transport; D051381:Rats
D007454:Iodides; D008164:Luminescent Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator