Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR

Area of Science:

• Biochemistry
• Genetics
• Pharmacology

Background:

• Cystic Fibrosis (CF) arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, impacting ion transport and anion homeostasis.
• The G542X allele, present in about 5% of CF patients, causes a premature stop codon, hindering the synthesis of functional CFTR protein.
• Developing therapeutic strategies to promote the readthrough of premature termination codons (PTCs) is a critical area for CF drug discovery.

Purpose of the Study:

• To establish and validate a high-throughput screening (HTS) assay for identifying small-molecule compounds that promote translational readthrough of the G542X-CFTR mutation.
• To discover novel modulators capable of restoring the synthesis of functional CFTR protein in CF patients carrying the G542X allele.

Main Methods:

• Implementation of a homogeneous, 1536-well functional G542X-CFTR readthrough assay using engineered HEK 293 cells.
• Monitoring of G542X-CFTR functional activity via membrane potential dye changes in response to cAMP stimulation and genistein potentiation.
• Automated screening of approximately 666,000 compounds, followed by secondary and tertiary assays for hit confirmation.

Main Results:

• The assay demonstrated robust performance with a Z' value of 0.69 ± 0.06, indicating suitability for HTS.
• Initial screening identified 7761 compounds, which were further refined through subsequent assays.
• A total of 188 confirmed hit compounds with low and submicromolar potencies were identified as G542X-CFTR readthrough modulators.

Conclusions:

• The developed phenotypic HTS assay is highly scalable and effective for discovering novel small-molecule G542X-CFTR readthrough modulators.
• This approach offers a promising strategy for developing new therapies targeting CF caused by premature termination codons.