Synthesis, biophysical, and pharmacological evaluation of the melanocortin agonist AST3-88: modifications of peptide backbone at Trp 7 position lead to a potent, selective, and stable ligand of the melanocortin 4 receptor (MC4R)

ACS Chem Neurosci. 2014 Oct 15;5(10):1020-31. doi: 10.1021/cn5000953. Epub 2014 Aug 26.


The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp(7) residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D (1)H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands.

Keywords: AGRP; GPCR; MC3R; MC4R; Melanotropin; agouti related protein; feeding behavior; feeding studies; obesity; peptide; reverse turn mimetic; serum stability; small molecule; solid phase synthesis.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Chromatography, Liquid
  • Drug Evaluation, Preclinical
  • Eating / drug effects
  • HEK293 Cells
  • Humans
  • Male
  • Mass Spectrometry
  • Mice
  • Molecular Dynamics Simulation
  • Molecular Structure
  • Neurotransmitter Agents / chemical synthesis
  • Neurotransmitter Agents / pharmacology*
  • Peptides, Cyclic / chemical synthesis
  • Peptides, Cyclic / pharmacology*
  • Proton Magnetic Resonance Spectroscopy
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Melanocortin, Type 3 / agonists
  • Receptor, Melanocortin, Type 3 / metabolism
  • Receptor, Melanocortin, Type 4 / agonists*
  • Receptor, Melanocortin, Type 4 / genetics
  • Receptor, Melanocortin, Type 4 / metabolism
  • Transfection
  • alpha-MSH / metabolism


  • AST3-88 peptide
  • MC4R protein, mouse
  • Mc3r protein, mouse
  • Neurotransmitter Agents
  • Peptides, Cyclic
  • Receptor, Melanocortin, Type 3
  • Receptor, Melanocortin, Type 4
  • alpha-MSH