Category: Automation and High-Throughput Technologies
Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance via anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy for identifying CFTR modulators utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. Here, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a bronchial epithelial cell line (CFBE41o-), which was engineered to report CFTR-mediated intracellular flux of iodide via a halide-sensitive YFP protein reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z’ value interval of 0.61±0.05. As further evidence of HTS suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays we identified 187 potential CFTR modulators, which EC50’s < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high throughput scalability to discover new small molecule CFTR modulators.
Emery Smith– Senior Associate, The Scripps Research Institute, Jupiter, FL
The Scripps Research Institute
Emery Smith is a Senior Associate at the Scripps Research Institute Molecular Screening Center located in Jupiter, FL, USA. Since joining the group in 2010, Emery has interacted both with corporate and academic partners to implement miniaturized, ultra-high-throughput screens of collections of up to 1 million compounds. The screens have been focused around GPCRs. Prior to joining the group in 2010, Emery was working with Dr. Charles Weissmann at Scripps working on Prion disease. This work started in 2006. Prior to 2006, Emery began his research career as a Research Associate at Athersys Inc. located in Cleveland, OH, USA. There he worked in both the VT (validated targets) group and the Regenerative Medicine group. The focus of this work was in the isolation and validation of adult human stem cells, currently known as Multistem.