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Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Published on: February 11, 2017
A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator
Vedrana Bali1, Ahmed Lazrak1, Purushotham Guroji1
1*Department of Cell, Developmental, and Integrative Biology, Department of Anesthesiology and Perioperative Medicine, The Lung Injury and Repair Research Center, and The Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Abstract:
Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.
Insights
Synonymous mutations in cystic fibrosis transmembrane conductance regulator (CFTR) can alter drug sensitivity. Combining corrector 4a (C4) and lumacaftor (VX-809) enhanced correction of ΔF508 CFTR variants, suggesting specific binding mechanisms.
Area of Science:
- Molecular biology
- Biochemistry
- Genetics
Background:
- The most common cystic fibrosis mutation, ΔF508 CFTR, involves deletion of Phe508.
- Synonymous mutations can influence protein biogenesis, structure, and function, potentially altering disease phenotype.
- Small-molecule drugs are being developed to correct ΔF508 CFTR function.
Purpose of the Study:
- To investigate the effects of distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on the biogenesis and function of ΔF508 CFTR variants with synonymous mutations (I507-ATT and I507-ATC).
- To understand the mechanisms of CFTR corrector drugs and the impact of synonymous mutations on drug sensitivity.
Main Methods:
- Analysis of ΔF508 CFTR variants (I507-ATT and I507-ATC) treated with C4 and/or VX-809.
- Biochemical assessment of protein correction and stability (Band B).
- Functional assessment using whole-cell currents activated by forskolin and IBMX.
Main Results:
- C4 alone stabilized I507-ATT ΔF508 CFTR but showed limited correction.
- VX-809 provided modest biochemical correction (~10%) and functional rescue for both variants, with evidence of posttranslational stabilization.
- Combination therapy (C4+VX-809) significantly enhanced correction and function (2-fold increase) for both I507-ATT and I507-ATC ΔF508 CFTR compared to VX-809 alone.
- C4 showed an additive effect with VX-809 on I507-ATC, suggesting it acts on VX-809-modified CFTR.
Conclusions:
- The binding of C4 and VX-809 to ΔF508 CFTR is conformation-specific.
- Synonymous mutations can indeed alter the sensitivity of CFTR variants to corrector drugs.
- Combined therapy offers enhanced therapeutic potential for specific CFTR mutations.
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