The ghrelin receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of β-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the ghrelin receptor. Moreover, mutagenesis studies of the receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.

]substance P, a series of novel, small, peptidemimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nM agonism potency was obtained and also in one case (wFw-Isn-NH 2 , where Isn is isonipecotic acid) ϳ80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH 2 was found to be a functionally biased agonist. Thus, wFw-Isn-NH 2 mediated potent and efficacious signaling through the G␣ q and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the G␣ 12/13 pathway. The recognition pattern of wFw-Isn-NH 2 with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH 2 was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH 2 binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH 2 is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.Ghrelin is a neuroendocrine hormone that differs from other peptide hormones by a fatty acid modification, which is crucial for both the binding and activation of its receptor (1). Ghrelin is synthesized mainly in the gastrointestinal tract, where the gene coding for the peptide sequence is expressed together with the enzyme responsible for the acylation of the fatty acid to the ghrelin peptide sequence (2, 3). Multiple functions have been described for ghrelin since it was discovered. Initially it was believed that growth hormone secretion induced by ghrelin receptors in the hypothalamus and the pituitary was the primary function of ghrelin (4). However, the function of ghrelin in the hypothalamus, and in particular in the arcuate nucleus, has become the focus of attention over the last decade. In the arcuate nucleus, ghrelin is responsible for increased activity in the NPY (neuropeptide Y) and AGRP (Agouti-related protein) neurones leading to increased appetite, decreased energy expenditure, and fat accumulation (5, 6). High receptor expression is also observed in the ventromedial nucleus of the hypothalamus, and the function in this area has been proposed to be orexigenic based on the regulation of fatty acid metabolism (7). More recently it has been demonstrated that ghrelin is also involved in reward-seeking behavior such as alcohol and ...

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