title: Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating.
abstract: Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.forename: Anang A
authors.initials: AA
authors.lastname: Shelat
authors.forename: R Kiplin
authors.initials: RK
authors.lastname: Guy
authors.forename: Vadiraj S
authors.initials: VS
authors.lastname: Gopinath
authors.forename: Tonghui
authors.initials: T
authors.lastname: Ma
authors.forename: Kai
authors.initials: K
authors.lastname: Du
authors.forename: Gergely L
authors.initials: GL
authors.lastname: Lukacs
authors.forename: Alessandro
authors.initials: A
authors.lastname: Taddei
authors.forename: Chiara
authors.initials: C
authors.lastname: Folli
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Pedemonte
authors.forename: Luis J V
authors.initials: LJ
authors.lastname: Galietta
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics* / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007454:Iodides / Q000493:pharmacokinetics*; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D010649:Phenylalanine; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000503:physiopathology*; D017384:Sequence Deletion; D013816:Thermodynamics; D013961:Thyroid Gland; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D007454:Iodides; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D010649:Phenylalanine; D019833:Genistein