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Research ArticleArticle

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

Yohei Norimatsu, Anthony Ivetac, Christopher Alexander, Nicolette O'Donnell, Leah Frye, Mark S. P. Sansom and David C. Dawson
Molecular Pharmacology December 2012, 82 (6) 1042-1055; DOI: https://doi.org/10.1124/mol.112.080267
Yohei Norimatsu
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Anthony Ivetac
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Christopher Alexander
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Nicolette O'Donnell
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Leah Frye
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Mark S. P. Sansom
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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David C. Dawson
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon (Y.N., C.A., N.O., D.C.D.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (A.I., M.S.P.S.); and Schrödinger Inc., Portland, Oregon (L.F.)
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Abstract

High-throughput screening has led to the identification of small-molecule blockers of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, but the structural basis of blocker binding remains to be defined. We developed molecular models of the CFTR channel on the basis of homology to the bacterial transporter Sav1866, which could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker such as N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101), which is thought to act by entering the channel from the extracellular side. As a more-stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual, ligand-docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest-scoring docked position was near two pore-lining residues, Phe337 and Thr338, and the rates of reactions of anionic, thiol-directed reagents with cysteines substituted at these positions were slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (Phe342) was replaced with alanine, the apparent affinity of the blocker was increased ∼200-fold. A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK45880]; the Cystic Fibrosis Foundation [Grant DAWSON08G0]; and the American Lung Association [Grant RT-7962-N]. The laboratory of M.S.P.S. was supported by the Wellcome Trust and the Biotechnology and Biological Sciences Research Council.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.080267.

  • ABBREVIATIONS:

    CFTR
    cystic fibrosis transmembrane conductance regulator
    2-ME
    2-mercaptoethanol
    DTT
    dithiothreitol
    MD
    molecular dynamics
    IFD
    induced-fit docking
    EC50(0)
    EC50 at 0 mV
    MM-GB/SA
    molecular mechanics-generalized Born/surface area
    IBMX
    3-isobutyl-1-methylxanthine
    MTSET+
    [2-(trimethylammonium)ethyl]methanethiosulfonate
    MTSES−
    [2-sulfonatoethyl]methanethiosulfonate
    NEM
    N-ethyl maleimide
    wt
    wild-type
    PDB
    Protein Data Bank
    TM
    transmembrane segment
    GlyH-101
    N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide
    iOWH032
    3-(3,5-dibromo-4-hydroxyphenyl)-N-(4-phenoxybenzyl)-1,2,4-oxadiazole-5-carboxamide.

  • Received May 28, 2012.
  • Accepted August 24, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (6)
Molecular Pharmacology
Vol. 82, Issue 6
1 Dec 2012
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Research ArticleArticle

Identification of a Binding Site for GlyH-101

Yohei Norimatsu, Anthony Ivetac, Christopher Alexander, Nicolette O'Donnell, Leah Frye, Mark S. P. Sansom and David C. Dawson
Molecular Pharmacology December 1, 2012, 82 (6) 1042-1055; DOI: https://doi.org/10.1124/mol.112.080267

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Research ArticleArticle

Identification of a Binding Site for GlyH-101

Yohei Norimatsu, Anthony Ivetac, Christopher Alexander, Nicolette O'Donnell, Leah Frye, Mark S. P. Sansom and David C. Dawson
Molecular Pharmacology December 1, 2012, 82 (6) 1042-1055; DOI: https://doi.org/10.1124/mol.112.080267
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