title: Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP.
abstract: We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.).
authors.forename: Yonjung
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.).
authors.forename: Marc O
authors.initials: MO
authors.lastname: Anderson
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.).
authors.forename: Jinhong
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.).
authors.forename: Min Goo
authors.initials: MG
authors.lastname: Lee
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.).
authors.forename: Wan
authors.initials: W
authors.lastname: Namkung
authors.affiliation: Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.) Alan.Verkman@ucsf.edu.
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C505032:CFTR protein, human; D010078:Oxazines; D011743:Pyrimidines; C474393:benzopyridopyrimidine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate