Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor

Abstract

ABSTRACT[D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]Substance P functions as a lowpotency 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 ligandbinding 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 structurefunctional 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.

Overlapping Binding Site for the Endogenous Agonist, Small-Molecule Agonists, and Ago-allosteric Modulators on the Ghrelin Receptor

“…The interaction with GluIII:09 was described very early on for MK-677 and later also for ghrelin (Feighner et al, 1998;Holst et al, 2006) just as the corresponding GluIII:09 (Glu 119 ) in the closely related motilin receptor has been shown to be a key charge-charge interaction point for small-molecule agonists-such as erythromycin (Xu et al, 2005).…”

Heterogeneity of Ghrelin/Growth Hormone Secretagogue Receptors

“…An inverse agonist for GHS-R1a is the synthetic D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P, a substance P and bombesin antagonist [129]; this is able to reduce constitutive signalling of GHS-R1a overexpressed in COS-7 cells [130, 131], but an endogenous counterpart remains to be identified. Most recently, shorter peptides derived from D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P were shown to display inverse agonist activity on GHS-R1a as well [131]. Finally, GHS-R1a antagonists are also available, such as D -Lys 3 -GHRP-6, L765-867 [6, 7, 120], isoxazole, diaminopyrimidine and triazole derivatives [132,133,134].…”

“…Hence the intracellular end of TM6 and TM7 move away from the center of the receptor toward TM3, exposing the sites subsequently recognized by G-protein and β-arrestin [137]. The ‘toggle switch model’, as it is known, is applicable to GHS-R1a, with a binding domain for the natural ligand ghrelin involving six amino acids located in TM3, TM6 or TM7 [131]. According to Pedretti et al [138], ligand binding and activation of GHS-R1a by ghrelin requires the ligand to interact with one pocket formed by polar amino acids and one formed by non-polar amino acids found in TM2/TM3 and TM5/TM6, respectively.…”

“…Recently, a synthetic CST-derived octapeptide, CST-8, which does not bind to SRIH-Rs, was shown to interact with GHS-R and to remove the inhibitory action of ghrelin on gastric acid volume and acid output [107]. An inverse agonist for GHS-R1a is the synthetic D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P, a substance P and bombesin antagonist [129]; this is able to reduce constitutive signalling of GHS-R1a overexpressed in COS-7 cells [130, 131], but an endogenous counterpart remains to be identified. Most recently, shorter peptides derived from D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P were shown to display inverse agonist activity on GHS-R1a as well [131].…”

Ghrelin – Physiological Functions and Regulation

“…113 However, studies in this area show conflicting results as ghrelin antagonist reduced bodyweight and food intake in some studies, 114,115 while it increased weight gain and food intake in another studies. 116,117 [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P was identified as an inverse agonist on GHS-R1a, 118 and several classes of ghrelin receptor antagonists have been developed (reviewed in reference 119).…”