Received for publication March 20, 2006.
Revised May 28, 2006.
Accepted for publication May 30, 2006.

GHRELIN RECEPTOR INVERSE AGONISTS - IDENTIFICATION OF AN ACTIVE PEPTIDE CORE AND ITS INTERACTION EPITOPES ON THE RECEPTOR

Abstract

[D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]-substance P functions as a low potency antagonist but a high potency 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 however in it self 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 TM-III, -VI and -VII. In contrast the inverse agonist peptides bind in a pocket, which extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI / -VII over 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, -IV and -V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deep across the main ligand-binding pocket and sterically blocking the movement of TM-VI and -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 being dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also non-peptide inverse agonists for the ghrelin receptor.

Key words: Neuropeptides, Gq/11 family, IP3/DAG, Func. analysis receptor/ion channel mutants, Receptor binding studies

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