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Molecular Pharmacology

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Research ArticleArticle

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Anthony L. Albiston, Vi Pham, Siying Ye, Leelee Ng, Rebecca A. Lew, Philip E. Thompson, Jessica K. Holien, Craig J. Morton, Michael W. Parker and Siew Yeen Chai
Molecular Pharmacology October 2010, 78 (4) 600-607; DOI: https://doi.org/10.1124/mol.110.065458
Anthony L. Albiston
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Vi Pham
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Siying Ye
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Leelee Ng
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Rebecca A. Lew
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Philip E. Thompson
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Jessica K. Holien
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Craig J. Morton
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Michael W. Parker
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Siew Yeen Chai
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Abstract

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn2+ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Footnotes

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

  • This research was supported by the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation; the Alzheimer's Drug Discovery Foundation; a Neurosciences Victoria/Victorian State Government Science, Technology, and Innovation Grant; the National Health and Medical Research Council (NHMRC) [Development Grants 454714 and 520695]; an NHMRC Peter Doherty fellowship (to V.P.); an Australian Research Council Federation Fellowship (to M.W.P.); an NHMRC Honorary Fellowship (to M.W.P.); and an NHMRC Senior Research Fellowship (to S.Y.C.).

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

    doi:10.1124/mol.110.065458.

  • ABBREVIATIONS:

    IRAP
    insulin-regulated aminopeptidase
    APN
    aminopeptidase N
    Ang IV
    angiotensin IV
    AVP
    arginine8 vasopressin
    HEK
    human embryonic kidney
    HFI
    Howard Florey Institute
    HFI-142
    ethyl 2-amino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate
    HFI-419
    ethyl 2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate
    HFI-435
    ethyl 2-amino-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
    HFI-437
    ethyl 2-acetylamino-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
    Leu-MCA
    l-leucine-4-methyl-7-coumarinylamide
    Leu-Enk
    leu-enkephalin
    LTA4H
    leukotriene A4 hydrolase
    LVV-H7
    Leu-Val-Val-hemorphin-7
    MCA
    7-amino-4-methylcoumarin
    TFA
    trifluoroacetic acid.

  • Received April 7, 2010.
  • Accepted July 2, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (4)
Molecular Pharmacology
Vol. 78, Issue 4
1 Oct 2010
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Research ArticleArticle

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Anthony L. Albiston, Vi Pham, Siying Ye, Leelee Ng, Rebecca A. Lew, Philip E. Thompson, Jessica K. Holien, Craig J. Morton, Michael W. Parker and Siew Yeen Chai
Molecular Pharmacology October 1, 2010, 78 (4) 600-607; DOI: https://doi.org/10.1124/mol.110.065458

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Research ArticleArticle

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Anthony L. Albiston, Vi Pham, Siying Ye, Leelee Ng, Rebecca A. Lew, Philip E. Thompson, Jessica K. Holien, Craig J. Morton, Michael W. Parker and Siew Yeen Chai
Molecular Pharmacology October 1, 2010, 78 (4) 600-607; DOI: https://doi.org/10.1124/mol.110.065458
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