Cystic fibrosis (CF) is a disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel. Mutations are classified into six classes with phenotypes from no CFTR protein synthesis to misfolding and/or functional defects.
Over the past seven years the FDA approved several novel small molecules that partially correct defects of different mutation classes of CFTR. This has triggered broad efforts to find better and/or different small molecule modulators that address even more CF disease-causing mutations. Here we present screening assays for two classes of CFTR variants: (1) F508del (causing protein misfolding and severely impaired cellular trafficking) and (2) premature termination codon (PTC) mutations, resulting in stop codons in the open reading frame of CFTR and no functional expression. Assays need to address the primary defects of these specific mutation types. A differential screening approach allows the discovery of class-specific hit molecules.
CFTR F508del leads to (1) misfolding of the nucleotide-binding domain 1 (NBD1) of CFTR and (2) perturbs normal interdomain interaction in the CFTR protein. An efficient therapy needs to address both protein folding defects for CFTR for rescue of CFTR functional expression. Suppressing one defect may allow identification of modulators of the 2nd defect. Thus, using specific suppressor mutations (R555K to restore NBD1 folding or R1070W to rescue domain-domain interactions, allelic screens were developed to enrich for small molecules that preferentially modulate interdomain interactions or NBD1 folding, respectively. The phenotypic screen relies on mammalian cells expressing CFTR F508del with the suppressor mutations and a reporter gene fused into an extracellular loop of CFTR. Hits from the two assays were further tested for complementary effects on trafficking rescue of CFTR F508del.
A different class of CFTR mutations are PTC variants (about 170 reported) that cannot be treated with available medicines. During CFTR protein synthesis, interaction of the ribosome with the PTC (UAA, UAG, or UGA) terminates protein translation. Furthermore, when the ribosome stalls at a PTC, translation-coupled RNA surveillance triggers the nonsense-mediated mRNA decay (NMD) pathway, resulting in a reduction of CFTR mRNA levels. Therefore, an effective therapy for CFTR PTC variants needs to address both premature translation termination and reduced CFTR mRNA. Cell-based assays to assess translational readthrough of PTCs have been developed based on either a reporter or the native CFTR gene. RT-qPCR of CFTR mRNA is utilized to monitor anti-NMD effects. Our data support the concept that combining readthrough modulators and NMD inhibitors may lead to more effective therapy.
The CF phenotypes for the above two classes of CFTR mutations derive from defects in different stages of CFTR biogenesis. Specific types of mutations require different screens for the identification of mutation class-specific disease modulators.