Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor

Birgitte Holst; Manja Lang; Erik Brandt; Anders Bach; Andrew Howard; Thomas M Frimurer; Annette Beck-Sickinger; Thue W Schwartz

doi:10.1124/mol.106.024422

Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor

Birgitte Holst, Manja Lang, Erik Brandt, Anders Bach, Andrew Howard, Thomas M Frimurer, Annette Beck-Sickinger, Thue W Schwartz

Department of Neuroscience

77 Citations (Scopus)

Abstract

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the ghrelin receptor.

Original language	English
Journal	Molecular Pharmacology
Volume	70
Issue number	3
Pages (from-to)	936-46
Number of pages	10
ISSN	0026-895X
DOIs	https://doi.org/10.1124/mol.106.024422
Publication status	Published - 2006

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@article{21c223e0f2fa11ddbf70000ea68e967b,

title = "Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor",

abstract = "[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the ghrelin receptor.",

author = "Birgitte Holst and Manja Lang and Erik Brandt and Anders Bach and Andrew Howard and Frimurer, {Thomas M} and Annette Beck-Sickinger and Schwartz, {Thue W}",

note = "Keywords: Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; COS Cells; Cells, Cultured; Cercopithecus aethiops; Epitopes; Ghrelin; Humans; Ligands; Models, Molecular; Molecular Sequence Data; Mutant Proteins; Peptide Hormones; Peptides; Protein Binding; Receptors, G-Protein-Coupled; Receptors, Ghrelin; Structure-Activity Relationship; Substance P",

year = "2006",

doi = "10.1124/mol.106.024422",

language = "English",

volume = "70",

pages = "936--46",

journal = "Molecular Pharmacology",

issn = "0026-895X",

publisher = "American Society for Pharmacology and Experimental Therapeutics",

number = "3",

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TY - JOUR

T1 - Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor

AU - Holst, Birgitte

AU - Lang, Manja

AU - Brandt, Erik

AU - Bach, Anders

AU - Howard, Andrew

AU - Frimurer, Thomas M

AU - Beck-Sickinger, Annette

AU - Schwartz, Thue W

N1 - Keywords: Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; COS Cells; Cells, Cultured; Cercopithecus aethiops; Epitopes; Ghrelin; Humans; Ligands; Models, Molecular; Molecular Sequence Data; Mutant Proteins; Peptide Hormones; Peptides; Protein Binding; Receptors, G-Protein-Coupled; Receptors, Ghrelin; Structure-Activity Relationship; Substance P

PY - 2006

Y1 - 2006

N2 - [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the ghrelin receptor.

AB - [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the ghrelin receptor.

U2 - 10.1124/mol.106.024422

DO - 10.1124/mol.106.024422

M3 - Journal article

C2 - 16798937

SN - 0026-895X

VL - 70

SP - 936

EP - 946

JO - Molecular Pharmacology

JF - Molecular Pharmacology

IS - 3

ER -

Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor

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