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Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor

Laboratory for Molecular Pharmacology, the Panum Institute, University of Copenhagen, Copenhagen, Denmark (B.H., E.B., A.B., T.W.S.); Institute of Biochemistry, Faculty of Bioscience, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany (M.L., A.B.-S.); Merck Research Laboratories, Rahway, New Jersey (A.H.); and 7TM Pharma A/S, Hørsholm, Denmark (T.M.F., T.W.S.)

[D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for 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

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