Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators.

Abstract

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

• Authors: Louise C Pyle, Annette Ehrhardt, Lisa High Mitchell, Lijuan Fan, Aixia Ren, Anjaparavanda P Naren, Yao Li, J P Clancy, Graeme B Bolger, Eric J Sorscher, Steven M Rowe
• Journal: American journal of physiology. Lung cellular and molecular physiology (Am J Physiol Lung Cell Mol Physiol) Vol. 301 Issue 4 Pg. L587-97 (Oct 2011) ISSN: 1522-1504 [Electronic] United States
• PMID: 21724857 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Aminophenols
• Cresols
• Membrane Transport Modulators
• Pyrazoles
• Quinolones
• VRT 532
• Cystic Fibrosis Transmembrane Conductance Regulator
• Colforsin
• ivacaftor

Topics

• Aminophenols (pharmacology)
• Animals
• Blotting, Western
• Cell Line
• Chlorocebus aethiops
• Colforsin (pharmacology)
• Cresols (pharmacology)
• Cystic Fibrosis (drug therapy, genetics, metabolism, pathology)
• Cystic Fibrosis Transmembrane Conductance Regulator (genetics, metabolism)
• Diffusion Chambers, Culture
• Dimerization
• Enzyme-Linked Immunosorbent Assay
• Gene Expression
• High-Throughput Screening Assays
• Humans
• Ion Channel Gating (drug effects)
• Ion Transport (drug effects)
• Lentivirus
• Membrane Transport Modulators (pharmacology)
• Mice
• Mutation
• Phosphorylation (drug effects)
• Protein Structure, Tertiary (genetics)
• Pyrazoles (pharmacology)
• Quinolones (pharmacology)
• Retroviridae
• Transduction, Genetic