Subtle changes in the sequence at the N-terminus and in the aromatic core of hexapeptidic ghrelin receptor inverse agonists can switch behavior from inverse agonism to agonism, but the C-terminal role of the sequence is unclear. Thus, analogs of the ghrelin receptor inverse agonist KbFwLL-NH2 (b = β-(3-benzothienyl)-d-alanine) were synthesized by solid phase peptide synthesis in order to identify the influence of aromaticity, charge, and hydrophobicity. Potency and efficacy of the hexapeptides were evaluated in inositol triphosphate turnover assays. Notably, modifications directly at the C-terminal Leu(6) could influence peptide efficacy leading to decreased constitutive activity. High hydrophobicity at the C-terminal position was of importance for elevated inverse agonist activity, the introduction of charged amino acids led to decreased potency. In contrast, structure-activity relationship studies of Leu(5) located closer to the aromatic core revealed an agonism-inducing position. These findings imply that amino acids with possible cation-π or π-π interactions and a suitable orientation at the C-terminus of the aromatic core induce agonism. Receptor binding studies showed that most peptides bind to the receptor at a concentration of 1 µM and modification directly at the C-terminus is generally more accepted than Leu(5) substitution. Interestingly, this observation is not dependent on the type of modification. These studies reveal another switch region of the short ghrelin receptor ligand pointing out the sensitivity of the ghrelin receptor binding pocket.
