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Molecular Pharmacology

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Research ArticleArticle

Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

Pari Malherbe, Olivier Roche, Anne Marcuz, Claudia Kratzeisen, Joseph G. Wettstein and Caterina Bissantz
Molecular Pharmacology July 2010, 78 (1) 81-93; DOI: https://doi.org/10.1124/mol.110.064584
Pari Malherbe
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Olivier Roche
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Anne Marcuz
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Claudia Kratzeisen
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Joseph G. Wettstein
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Caterina Bissantz
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Abstract

The orexins and their receptors are involved in the regulation of arousal and sleep–wake cycle. Clinical investigation with almorexant has indicated that this dual OX antagonist is efficacious in inducing and maintaining sleep. Using site-directed mutagenesis, β2-adrenergic-based OX1 and OX2 modeling, we have determined important molecular determinants of the ligand-binding pocket of OX1 and OX2. The conserved residues Asp45.51, Trp45.54, Tyr5.38, Phe5.42, Tyr5.47, Tyr6.48, and His7.39 were found to be contributing to both orexin-A-binding sites at OX1 and OX2. Among these critical residues, five (positions 45.51, 45.54, 5.38, 5.42, and 7.39) were located on the C-terminal strand of the second extracellular loop (ECL2b) and in the top of TM domains at the interface to the main binding crevice, thereby suggesting superficial OX receptor interactions of orexin-A. We found that the mutations W214A45.54, Y223A5.38, F227A5.42, Y317A6.48, and H350A7.39 resulted in the complete loss of both [3H]almorexant and [3H]N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA) binding affinities and also blocked their inhibition of orexin-A-evoked [Ca2+]i response at OX2. The crucial residues Gln1263.32, Ala1273.33, Trp20645.54, Tyr2155.38, Phe2195.42, and His3447.39 are shared between almorexant and 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042) binding sites in OX1. The nonconserved residue at position 3.33 of orexin receptors was identified as occupying a critical position that must be involved in subtype selectivity and also in differentiating two different antagonists for the same receptor. In summary, despite high similarities in the ligand-binding pockets of OX1 and OX2 and numerous aromatic/hydrophobic interactions, the local conformation of helix positions 3.32, 3.33, and 3.36 in transmembrane domain 3 and 45.51 in ECL2b provide the structural basis for pharmacologic selectivity between OX1 and OX2.

Footnotes

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

    doi:10.1124/mol.110.064584.

  • ABBREVIATIONS:

    GPCR
    G-protein coupled receptor
    OX1
    orexin 1 receptor
    OX2
    orexin 2 receptor
    almorexant
    (2R)-2-{(1S)-6,7-dimethoxy-1-[2-(4-trifluoromethyl-phenyl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide (ACT-078573)
    EMPA
    N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide
    SB-674042
    1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone
    3D
    three-dimensional
    TM
    transmembrane
    7TMD
    seven-transmembrane domain
    [Ca2+]i
    cytosolic free Ca2+ concentration
    FLIPR
    fluorometric imaging plate reader
    AR
    adrenergic receptor
    WT
    wild type
    NK
    neurokinin receptor
    PDB
    Protein Data Bank
    BSA
    bovine serum albumin
    HEK
    human embryonic kidney
    ECL
    extracellular loop
    NSB
    nonspecific binding
    SB
    specific binding.

  • Received March 9, 2010.
  • Accepted April 16, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (1)
Molecular Pharmacology
Vol. 78, Issue 1
1 Jul 2010
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Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-p…
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Research ArticleArticle

Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

Pari Malherbe, Olivier Roche, Anne Marcuz, Claudia Kratzeisen, Joseph G. Wettstein and Caterina Bissantz
Molecular Pharmacology July 1, 2010, 78 (1) 81-93; DOI: https://doi.org/10.1124/mol.110.064584

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Research ArticleArticle

Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

Pari Malherbe, Olivier Roche, Anne Marcuz, Claudia Kratzeisen, Joseph G. Wettstein and Caterina Bissantz
Molecular Pharmacology July 1, 2010, 78 (1) 81-93; DOI: https://doi.org/10.1124/mol.110.064584
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