title: 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: Departments of Genetics, University of Alabama at Birmingham, 35294-0006, USA.
authors: Louise C
authors: LC
authors: Pyle
authors: Annette
authors: A
authors: Ehrhardt
authors: Lisa High
authors: LH
authors: Mitchell
authors: Lijuan
authors: L
authors: Fan
authors: Aixia
authors: A
authors: Ren
authors: Anjaparavanda P
authors: AP
authors: Naren
authors: Yao
authors: Y
authors: Li
authors: J P
authors: JP
authors: Clancy
authors: Graeme B
authors: GB
authors: Bolger
authors: Eric J
authors: EJ
authors: Sorscher
authors: Steven M
authors: SM
authors: Rowe
mesh_terms: D000627:Aminophenols / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D003408:Cresols / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D015194:Diffusion Chambers, Culture; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D016086:Lentivirus; D049990:Membrane Transport Modulators / Q000494:pharmacology*; D051379:Mice; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011720:Pyrazoles / Q000494:pharmacology; D015363:Quinolones / Q000494:pharmacology; D012190:Retroviridae; D014161:Transduction, Genetic
chemical_list: D000627:Aminophenols; D003408:Cresols; D049990:Membrane Transport Modulators; D011720:Pyrazoles; D015363:Quinolones; C535089:VRT 532; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C545203:ivacaftor