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

Laboratory for Molecular Pharmacology, University of Copenhagen, the Panum Institute, Copenhagen, Denmark (B.H., J.M., T.H., K.B.C., C.R.U., T.W.S.); and 27TM Pharma A/S, Hørsholm, Denmark (T.M.F., T.W.S.)

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.

Address correspondence to: Dr. Birgitte Holst, Laboratory for Molecular Pharmacology; The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. E-mail: b.holst{at}molpharm.dk