Drug Target Strategies
Protein Homeostasis: new drug discovery directions
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. Nearly 5% of CF patients possess one or more copies of the G542X, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small molecule modulators of mutant CFTR biosynthesis that affect “readthrough” of this stop codon, or premature termination codon (PTC) to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large scale screening of a homogeneous, miniaturized 1536-well functional G542X-CFTR readthrough assay. The assay utilizes HEK293 cells engineered to over-express the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR, such that compounds that allow translational readthrough and synthesis of functional CFTR chloride channels will be reflected by changes in membrane potential in response to cAMP stimulation with forskolin, and CFTR channel potentiation with genistein. Assay statistics were excellent with Z’ values of 0.69±0.06 despite a S:B of 1.19±0.04. As further evidence of HTS suitability, we completed automated screening of 666,120 compounds, identifying 7,761 initial hits. Following secondary and tertiary assays, we have identified 188 confirmed hit compounds with low and sub-micromolar potencies. Thus, the assay has integrated the advantages of a phenotypic screen with high throughput scalability to identify new small molecule G542X-CFTR readthrough modulators.