Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor.
[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.
Holst
Birgitte
B
Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. b.holst@molpharm.dk
Lang
Manja
M
Brandt
Erik
E
Bach
Anders
A
Howard
Andrew
A
Frimurer
Thomas M
TM
Beck-Sickinger
Annette
A
Schwartz
Thue W
TW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000939:Epitopes / Q000378:metabolism*; D054439:Ghrelin; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000819:agonists / Q000737:chemistry; D036361:Peptide Hormones / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000737:chemistry; D054440:Receptors, Ghrelin; D013329:Structure-Activity Relationship; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry
D000939:Epitopes; D054439:Ghrelin; D008024:Ligands; D050505:Mutant Proteins; D036361:Peptide Hormones; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D013373:Substance P; C045800:substance P, Phe(5)-Trp(7,9)-Leu(11)-