Determination of Unique AGRP and MC4R Interactions: Use of Stereochemical Modifications of Ligands and Receptor Mutagenesis

摘要

The melanocortin system belongs to a superfamily of GPCRs and consists of endogenous agonists, antagonists and five receptor subtypes [1]. The MC4R has been shown to be involved weight and energy homeostasis and feeding behavior [2]. MC4R deficient mice exhibit an obese and hyperphagic phenotype proposing that the MC4R is involved in the regulation of feeding behavior. [2] The antagonist, AGRP, is a target of interest because is involved in the regulation of food intake and is a competitive antagonist of the MC4R [3]. Therefore, it is postulated that AGRP mediates its effects through the MC4R in the CNS [1-3]. The C-terminus of AGRP is a cysteine-rich region of the protein and has receptor binding affinity and antagonism comparable to the full length protein. This region contains five disulfide bridges with a conserved Arg-PhePhe motif that has been identified as being important for the antagonistic activity of AGRP [45]. Truncation of this domain has led to the identification of monocyclic and bicyclic derivatives of AGRP that provide support to the hypothesis that not all five disulfide bridges are necessary for molecular recognition and receptor antagonism [6-8]. The stereochemical inversion of the Arg-Phe-Phe region led to the discovery of a new melanocortin agonist template [6]. Previous studies have shown that the incorporation of DArg~(111) in the monocyclic compound (EMH1-100) converts an antagonist into an agonist at the MC4R [6]. Within the homology model of the mMC4R complexed with hAGRP(87-132) [9], a putative interaction was observed that may explain the pharmacology [6]. It is postulated that DArg~(111) interacts with MC4R Asnl 15 [8]. Previous studies showed that the bicyclic peptide (EMH2-93) displayed equipotent binding affinity to that of hAGRP (87-132) at the mMC4R [7]. However, it was an 80-fold less potent antagonist at the mMC4R [7]. Homology modeling demonstrated a postulated interaction of Arg~(111) of the bicyclic compound interacting with Asnl 15 of mMC4R. This Arg~(111)-mMC4R Asnl 15 interaction was not observed for hAGRP (87-132) and may attribute to the change in antagonist potency observed for the bicyclic compound [8]. It is hypothesized that similar pharmacological results will be observed when Arg~(111) is stereochemically inverted in the bicyclic compound as seen with the monocyclic compounds.