A Novel Non-Peptidic Agonist of the Ghrelin Receptor with Orexigenic Activity In vivo

Abstract

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand.

Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists

“…For that reason, they display signaling pathway characteristics similar to those of the endogenous ligand, ghrelin. In addition, we showed that pyridone, a novel nonpeptide GHSR-1a agonist, recently identified in our laboratory (44), increases Ca 2+ influx (EC 50 , 3.1 mM), but is unable to regulate GHSR-1a internalization, even at 10 mM, nor does it increase b-arrestin-1 recruitment, suggesting a signaling bias and functional selectivity toward G-protein-dependent signaling.…”

“…To investigate the roles of these GHSR-1a ligands in b-arrestin-dependent signaling, b-arrestin-1 recruitment was analyzed in a U2OS cell line. Ghrelin, MK-0677, and L692,585 (500 nM each) showed a classic agonist response increasing b-arrestin-1 recruitment; however, this effect was not observed after treatment with 500 nM pyridone, which had been identified as a novel nonpeptide GHSR-1a agonist in our laboratory (44) (Fig. 2B).…”

“…Several synthetic compounds have been described in the literature as GHSR-1a ligands. These compounds have been categorized as agonists, antagonists, and inverse agonists; however, recent evidence has suggested that the specific GHSR-1a downstream signaling pathways modulated by GHSR-1a ligands display signaling bias (37,(41)(42)(43)(44), meaning that certain ligands can differentially modulate G-protein-dependent vs. G-protein-independent signaling, which is likely to result in differential functional and behavioral responses (45). Indeed, the concepts of functional selectivity and ligand bias are becoming increasingly appreciated in GPCR drug discovery (46).…”

A Dairy-Derived Ghrelinergic Hydrolysate Modulates Food Intake In Vivo

“…GHSR-1a mediated changes in intracellular Ca 2+ were recorded on a High-Throughput Cellular Screening System (Molecular Devices Corporation, Sunnyvale, San Jose, CA, USA). Ca 2+ mobilisation assays were performed according to a protocol modified from a previously described method [ 39 ]. Stably transfected human embryonic kidney (HEK293A) cells overexpressing GHSR-1a were seeded in sterile 96-well microtiter plates with black-walled and clear-bottomed wells (3904, Costar, Fisher Scientific, Dublin, Ireland) at a density of 2.5 × 10 4 cells per well.…”

“…Calcium imaging took place for HEK-GHSR-1a cells seeded on a 12-well plate at 2.0 × 10 5 cells/mL two days before the assay according to a previously described method [ 39 ]. The day before the assay, media was swapped to serum-free.…”