New Trisubstituted 1,2,4-Triazole Derivatives as Potent Ghrelin Receptor Antagonists. 3. Synthesis and Pharmacological in Vitro and in Vivo Evaluations

Abstract

Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the alpha-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

“…The amino acid sequences of Trp 3 -ghrelin 1-8 , desacyl-ghrelin 1-8 , and X 1-8 are described in (B). Additional symbols and abbreviations are defined as follows: T7prom, T7 promoter; Ω, translation enhancer of tobacco mosaic virus; Kozak, Kozak sequence for translation initiation; PDO and BDA, POU-specific DNA-binding domain of Oct-1 (PDO) or B domain of protein A (BDA) as a scaffold protein; 6xHis, hexahistidine tag; Xa, the recognition site for factor Xa protease; Trp 3 -ghrelin 1-8 , desacyl-ghrelin 1-8 ; X [1][2][3][4][5][6][7][8] , eight N-terminal amino acids of the ghrelin mutant or eight randomized amino acids; ghrelin 9-28 , 20 C-terminal amino acids of ghrelin; and LHR, the hybridization region for the puromycin linker. (B) Amino acid sequences of the regions of ghrelin used in the selection process.…”

“…Therefore, GHS-R antagonists are expected to perform antiobesity functions by suppressing food intake and weight gain. In fact, small-molecule GHS-R antagonists and [D-Lys-3]-GHRP-6, which is one of the few known peptide antagonists of GHS-R, decrease food intake and weight gain via peripheral administration (7)(8)(9)(10).…”

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

“…GSK161443, a small molecule ghrelin antagonist of calcium mobilization and inositol phosphate turnover (58), acts in vivo as an antagonist of GH release, an inducer of food intake, and a promoter of increased body weight (59,60), but the mechanistic and physiological basis for this has not been determined. Other described small molecule non-peptide agonists (34,61) and antagonists (36,62,63) of GHSR1a have been developed to treat GH deficiency, cachexia, and obesity, and, in a few cases, GH release was discriminated from food intake (59,62,63). ERK1/2 is required for the proliferative effect of ghrelin in intestinal epithelial and pancreatic endothelial cells (64 -66), and G protein activation and ␤-arrestin scaffolding are both associated with enhanced cell growth (1,64).…”

High Constitutive Activity Is an Intrinsic Feature of Ghrelin Receptor Protein

“…The protein mixture after lipid disc reconstitution was directly loaded on the column. After washing with 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, pH 7.4, the bound proteins were recovered by washing the column with the 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, buffer containing 0.1 mM JMV 4101, a low affinity antagonist (in the 0.1 M range) derived from JMV 3002 (37). The antagonist was then removed through extensive dialysis against a 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, pH 7.4, buffer.…”