A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists-the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5-yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide], the spiroindoline sulfonamide MK-677 [N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4'-piperidin)-1'-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide], and two novel oxindole derivatives, SM-130686 [(+)-6-carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole] and SM-157740 [(+/-)-6-carbamoyl-3-(2, 4-dichlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole)]. The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09-to-Gln substitution in the extracellular segment of TM-III. Likewise, all six agonists were affected by substitutions of PheVI:16, ArgVI:20, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists--including ago-allosteric modulators--and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor.

2005-09-01·Molecular endocrinology (Baltimore, Md.)

Nonpeptide and Peptide Growth Hormone Secretagogues Act Both as Ghrelin Receptor Agonist and as Positive or Negative Allosteric Modulators of Ghrelin Signaling

Article

作者: Heding, Anders ; Holst, Birgitte ; Schwartz, Thue W. ; Bach, Anders ; Brandt, Erik

Abstract:

Two nonpeptide (L692,429 and MK-677) and two peptide [GH-releasing peptide (GHRP)-6 and ghrelin] agonists were compared in binding and in signal transduction assays: calcium mobilization, inositol phosphate turnover, cAMP-responsive element (CRE), and serum-responsive element (SRE) controlled transcription, as well as arrestin mobilization. MK-677 acted as a simple agonist having an affinity of 6.5 nm and activated all signal transduction systems with similar high potency (0.2–1.4 nm). L-692,429 also displayed a very similar potency in all signaling assays (25–60 nm) but competed with a 1000-fold lower apparent affinity for ghrelin binding and surprisingly acted as a positive allosteric receptor modulator by increasing ghrelin’s potency 4- to 10-fold. In contrast, the potency of GHRP-6 varied 600-fold (0.1–61 nm) depending on the signal transduction assay, and it acted as a negative allosteric modulator of ghrelin signaling. Unexpectedly, the maximal signaling efficacy for ghrelin was increased above what was observed with the hormone itself during coadministration with the nonendogenous agonists. It is concluded that agonists for the ghrelin receptor vary both in respect of their intrinsic agonist properties and in their ability to modulate ghrelin signaling. A receptor model is presented wherein ghrelin normally only activates one receptor subunit in a dimer and where the smaller nonendogenous agonists bind in the other subunit to act both as coagonists and as either neutral (MK-677), positive (L-692,429), or negative (GHRP-6) modulators of ghrelin function. It is suggested that an optimal drug candidate could be an agonist that also is a positive modulator of ghrelin signaling.

2004-01-01·Bioorganic & medicinal chemistry letters4区 · 医学

Parallel solid-phase synthesis of disubstituted (5-biphenyltetrazolyl) hydantoins and thiohydantoins targeting the growth hormone secretagogue receptor

4区 · 医学

Article

作者: Jesper F. Lau ; Mikael Begtrup ; Rune Severinsen ; Michael Ankersen ; Kent Bondensgaard ; Birgit S. Hansen

An efficient solid-phase protocol for the synthesis of substituted (5-biphenyltetrazolyl)-hydantoins and -thiohydantoins has been developed. Suzuki cross-coupling between resin-bound 2-(tetrazol-5-yl)-phenylborinane and 4-bromobenzaldehyde gave the corresponding tetrazolylbiphenyl aldehyde. Subsequent reductive amination using amino acid esters gave the pivotal resin bound amino acid esters which were transformed to hydantoins or thiohydantoins via two routes: (i) treatment with isocyanates or isothiocyanates or (ii) successive treatment with triphosgene and primary amines. Using molecular modeling, we were able to jump from L-692,429, a well known non-peptidyl growth hormone secretagogue (GHS), to biphenyltetrazolyl hydantoins, obtaining compounds with IC(50) values below 600 nM after two iterative cycles only.

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