Abstract
[d-Arg1,d-Phe5,d-Trp7,9,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.
Footnotes
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This study was supported by grants from the Danish Medical Research Council, The Novo Nordisk Foundation, and The Weimann Foundation (to B.H.).
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ABBREVIATIONS: NPY, neuropeptide Y; AgRP, Agouti related peptide; 7TM, seven transmembrane; SAR, structure-activity relationship; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; MK-677, 2-amino-N-((R)-2-(benzyloxy)-1-((1-(methylsulfonyl)spiro(indoline-3,4′-piperidin)-1′-yl)carbonyl)ethyl)-2-methyl-propionamide monomethanesulfonate.
- Received March 16, 2006.
- Accepted May 30, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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