Overlapping Binding Site for the Endogenous Agonist, Small-Molecule Agonists, and Ago-allosteric Modulators on the Ghrelin Receptor

Abstract

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-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.The gastrointestinal peptide hormone ghrelin is an important regulator of appetite, energy expenditure, and acute growth hormone secretion through interaction with the ghrelin receptors located mainly in the central nervous system (Tschöp et al., 2000). From a drug discovery point of view, these functions of ghrelin qualify ghrelin receptor agonists as anabolic compounds with potentials for treatment of, for example, cachexia.The initial agonists synthesized for the ghrelin receptor were peptides, sharing common structural features, including a central hydrophobic motif and a positive charge in the amidated C-terminal end. Despite relatively poor bioavailability and low therapeutic index/window, these peptides efficiently induced GH secretion in vitro as well as in vivo both in animal and human models (Bowers et al., 1984). GHRP-6 is a prototype for these peptides (Fig. 1). In an attempt to increase oral bioavailability, a series of nonpeptide compounds was subsequently developed. They were

A novel truncated glucagon-like peptide 2 (GLP-2) as a tool for analyzing GLP-2 receptor agonists

“…Together, the results from the reporter and binding assays suggested that the oxime derivatives were bound to the allosteric site of human GLP-2R. It has been speculated that the binding sites of several ago-allosteric modulators overlap with those of the endogenous ligands, for example, ghrelin (13,21). However, it has not been clarified how an agoallosteric modulator binds when its binding site overlaps with that of an orthosteric ligand, although some scenarios have been presented to explain the phenomenon (21).…”

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

“…Previously described agonists for the ghrelin receptor (peptides and non-peptide ligands) are all strongly dependent on GluIII:09 as an anchor point (21,41). For example, the potency of ghrelin itself is decreased 250-fold, and the prototype smallmolecule agonist MK-677 is affected more than 10.000-fold by substitution at this position (Fig.…”

“…For example, the potency of ghrelin itself is decreased 250-fold, and the prototype smallmolecule agonist MK-677 is affected more than 10.000-fold by substitution at this position (Fig. 8, A and B) (21,41). GluIII:09 is thought to play a similar role for agonist ligands in the ghrelin receptor as the neighboring classical AspIII:08 does for ligands in general in the monoamine receptor system (42).…”

“…7A). The substitutions were selected from a library of mutants based on their properties in both introducing significant structural change and being expressed at a reasonable level at the cell surface compared with the wild-type receptor, as published previously (41). Thus, as judged by cell surface ELISA, the expression level of the mutants was between 0.37-and 1.3-fold of the expression level of the wild-type receptor ( Table 2).…”

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

“…In this respect, the ghrelin receptor is particularly suited because even in the absence of its endogenous agonist, it displays almost 50% of its maximal signaling capacity, and, importantly, the ghrelin receptor is generally robust with respect to mutational changes (20,21).…”