Two nonpeptide (L692,429 and MK-677) and two peptide [GH-releasing peptide (GHRP)-6 and ghrelin] agonists were compared in binding and in signal transduction assays: calcium mobilization, inositol phosphate turnover, cAMP-responsive element (CRE), and serum-responsive element (SRE) controlled transcription, as well as arrestin mobilization. MK-677 acted as a simple agonist having an affinity of 6.5 nm and activated all signal transduction systems with similar high potency (0.2-1.4 nm). L-692,429 also displayed a very similar potency in all signaling assays (25-60 nm) but competed with a 1000-fold lower apparent affinity for ghrelin binding and surprisingly acted as a positive allosteric receptor modulator by increasing ghrelin's potency 4- to 10-fold. In contrast, the potency of GHRP-6 varied 600-fold (0.1-61 nm) depending on the signal transduction assay, and it acted as a negative allosteric modulator of ghrelin signaling. Unexpectedly, the maximal signaling efficacy for ghrelin was increased above what was observed with the hormone itself during coadministration with the nonendogenous agonists. It is concluded that agonists for the ghrelin receptor vary both in respect of their intrinsic agonist properties and in their ability to modulate ghrelin signaling. A receptor model is presented wherein ghrelin normally only activates one receptor subunit in a dimer and where the smaller nonendogenous agonists bind in the other subunit to act both as coagonists and as either neutral (MK-677), positive (L-692,429), or negative (GHRP-6) modulators of ghrelin function. It is suggested that an optimal drug candidate could be an agonist that also is a positive modulator of ghrelin signaling.

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation. 7TM3 receptors (G-protein-coupled receptors) constitute one of the largest superfamilies of proteins, which also serve as targets for a large proportion of current medical drugs. In particular members of the family A or rhodopsin-like 7TM receptors, which also is the largest family, are considered to be rather easy drug targets. Most of these receptors are antagonist-prone, i.e. if they are screened with libraries of small organic, drug-like molecules, most if not all of the hits will be antagonists, inhibiting agonist-induced signal transduction, when they are tested in functional assays (1). However, a small proportion of the 7TM receptors, including, for example, the complement C5a receptor, the melanocortin MC4 receptor, and the ghrelin receptor, are instead agonist-prone, i.e. most of the screening hits are agonists in functional assays (2). Part of the reason for this is probably that these receptors are characterized by a rather high degree of constitutive, ligand-independent signaling activity, i.e. in the conformational equilibrium these receptors are at least partly biased for active conformation(s) (1). The ghrelin receptor (Fig. 1), for example, is among the most constitutively active receptors as it signals with ϳ50%, depending on the signal transduction pathway, of its maximal signaling capacity without the presence of any hormone (3, 4). High constitutive activity has b...

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.

Absorbable tack fixation of the mesh was associated with a higher risk of recurrence than non-absorbable tacks for laparoscopic mesh repair of incisional hernia, but did not influence chronic pain.

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