Research reportAssessment of a small molecule melanocortin-4 receptor-specific agonist on energy homeostasis
Introduction
The involvement of the MC4-R in the regulation of food intake has been demonstrated in a knockout mouse model [16]. MC4-deficient mice were found to develop maturity-onset obesity and treatment with melanocortin agonists had no effect on feeding [18]. Later, it was shown that MC4-R knockout mice exhibit deranged thermogenic and locomotor responses when the fat content of their food is elevated [5] indicating that, in addition to ingestive behavior, MC4-R activation modulates physiologic and behavioral responses to diet composition. The engineered deletion of the MC3-R gene demonstrated that the MC3-R is not required for the metabolic and locomotor response to nutrient content of the available diet [4], [6]. This knockout model, however, does point to a role for the MC3-R in directing energy partitioning [5].
Despite the wealth of information gleaned from these and other genetic models [24], our understanding of the role of the central melanocortin system in regulating feeding behavior has been limited by the lack of receptor-selective agonists and antagonists [3]. Previous pharmacological studies using melanocortin peptide derivatives (MTII and SHU9119, a broad agonist and antagonist, respectively) have not been useful in discerning the relative contributions of MC4-R and MC3-R to observed effects on feeding and weight control [9], [13]. An ideal drug candidate would be a low molecular weight compound selective for one melanocortin receptor subtype that mimicked the potency of the endogenous melanocortin ligands (α-MSH, γ-MSH, and AgRP) with no adverse effects on the animal model tested.
Researchers at Merck have recently reported the development of an orally active small molecule peptide mimetic MC4-R agonist [23]. This compound, 1,2,3R,4-tetrahydro-isoquinoline-3-carboxylic acid [1R-(4-chloro-benzyl)-2-(4-cyclohexyl-4-[1,2,4]triazol-1-ylmethyl-piperidin-1-yl)-2-oxo-ethyl]-amide, is a full agonist on the human and mouse MC4-R with both good specificity and potent in vitro activity. Although this new class of agonist was reported to decrease feeding following administration in animal models, the only in vivo biological data provided [26] relates to the MC4-R mediated stimulation of erectile function [28]. To better characterize its role in regulating feeding behavior, we synthesized this compound (Compound-1) by modification of the methods reported in the original patent, fully characterized it pharmacologically and determined its effectiveness in inhibiting feeding in rodents.
Section snippets
Synthesis of compound and validation
Compound-1 was synthesized with minor modifications according to the methods described by Sebhat et al. [26] from ethyl 4-phenyl-4-piperidinecarboxylate. The coupling of the free base, 4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine, with Boc-d-pCl-phenylalanine was accomplished with one equivalent of HBTU (2-(1H-benzotriazole-1-yl)-1,1,3tetramethyl) uranium hexaflurophosphate with two equivalents of diisopropyl N-ethyl amine. The method using EDC/HOBt with the TFA salt described in the
Synthesis and purification
As it was not possible to dissolve Compound-1 directly in aqueous solutions, compound was first dissolved in 100% dimethylsulfoxide (DMSO) then diluted to 10% DMSO with filtered water. The stability of the compound was tested by adding it to freshly harvested rat plasma, where it lost approximately 25% of its activity, by in vitro adenylyl cyclase assay, after 4 h at 37 °C, and where it lost less than 5% activity following a 3-day incubation at 37 °C in vehicle (data not shown).
Discussion
The development of efficacious therapeutics to treat human obesity is hindered by difficulties in engineering compounds capable of targeting a specific component of one of the seemingly myriad energy homeostasis pathways (for review see Bray and Greenway [3]). Here we confirm the in vitro potency and selectivity for a peptide mimetic MC4-R-small-molecule agonist and explore the ability of this compound to suppress feeding and affect metabolism in acute administration models [23], [26]. Using
Acknowledgements
The authors thank Victor Hruby for peptides and Heather Takahashi for help preparing the manuscript. This work was supported by NIH AR42415.
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