C‐terminus of a hexapeptidic ghrelin receptor inverse agonist can switch peptide behavior from inverse agonism to agonism

Abstract

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.

Development of Fluorinated Non-Peptidic Ghrelin Receptor Ligands for Potential Use in Molecular Imaging

“…Ghrelin served as control with an EC 50 of 1.0 nM (pEC 50 = 9.00 ± 0.05, E max = 100% ± 3%), which was comparable to data from previous experiments [49,50]. All compounds were found to be partial agonists at the GhrR with similar potencies in the low nanomolar range.…”

“…Unlabeled ghrelin served as control with an IC 50 of 1.7 nM, comparable to previous experiments [50]. ( S )- 9 was the starting compound for modification with an IC 50 of 2.2 nM (Figure 1A).…”

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

“…For the assay, COS‐7 cells stably expressing the ghrelin receptor were used, and after stimulation with the peptide, the intracellular generation of IPs by the Gα q/11 signaling pathway of the GhrR was determined. Measurements were normalized to the control peptide ghrelin, which possesses a high potency at the GhrR (EC 50 = 1 nM) in accordance with previous studies . Peptide 8 was found to be a super‐agonist for the GhrR with nanomolar potency and a higher maximal receptor activation (E max = 135%) compared with ghrelin.…”

Side Chain Orientation of Tryptophan Analogues Determines Agonism and Inverse Agonism in Short Ghrelin Peptides

“…However, the additional phenyl side chain may regain the lost activity. Leu 5 of KbFwLL‐NH 2 was recently replaced by amino acids with various characteristics and it was shown that Lys, Gln, Phe, and Ala can induce agonism . The most efficient agonist was the d ‐2Nal 5 analogue, whereas 2Nal 5 had no activity at the ghrelin receptor.…”

“…Interestingly, the d ‐2Nal 4 analogue 19 was 4‐fold more active than the Wrf 4 analogue ( 13 ), but the differences were diminished in the combined agonist. d ‐2Nal possesses strong agonism‐prone characteristics at position 4 and can even turn the efficient inverse agonist KbFwLL‐NH 2 ( 15 ) into an agonist by substitution of Leu 5 . It is surprising that 20 can almost reach the same activity as 18 .…”