An Aromatic Region To Induce a Switch between Agonism and Inverse Agonism at the Ghrelin Receptor

Abstract

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.

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling

“…Thanks to G protein BRET biosensors, we directly demonstrated that GHS-R1a constitutively activates both G q and G 13 . We found that K-(D-1Nal)-FwLL-NH 2 , which had been previously characterized as a GHS-R1a inverse agonist on the IP pathway (38), is indeed an efficient and potent inverse agonist toward G q activity but also behaves as an inverse agonist toward G 13 . We also found that SPA as well as KwFwLL-NH 2 and JMV 4484 behave as inverse agonists toward IP production and G q activation but were silent toward G 13 activation.…”

“…This chiral center contains an amino function, which was elongated by the Leu-Leu dipeptide, and a lysine residue was then introduced in the N-terminal part to mimic the peptide core of the substance P analog. Peptides KwFwLL-NH 2 and K-(D-1Nal)-FwLL-NH 2 were synthesized at Institut des Biomolécules Max Mousseron as described previously (37,38). The thromboxane A 2 receptor agonist U46619 was purchased from Cayman Chemical.…”

High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry

“…Addition of 750 ll dilution buffer and removal of cell debris was carried out before purification of the supernatant on an anion-exchange resin. 34,36,37 GraphPad Prism 5.0 (GraphPad Software, San Diego, USA) was used to analyze the data. For clarity, dpm values were normalized to the constitutive activity, that is, 100% of constitutive activity represents the activity of the receptor without the influence of peptides.…”

“…34 Human ghrelin served as a control. IC 50 values of the binding curves were obtained with GraphPad Prism 5.0.…”

Receptors of Peptides as Therapeutic Targets in Epilepsy Research

“…Recently, another interesting hypothesis was suggested based on the finding that the GHSR1a receptor has significant basal activity [224] and that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates [225]: an endogenous agonist can have anticonvulsant action by reducing the activity of the constitutively active receptor with a combination of inverse agonism and desensitization/internalization of the receptor [225]. This hypothesis should be tested by newly developed inverse agonists of the GHSR1a receptor [226,227].…”