title: 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).
abstract: 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.
authors.affiliation: Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida , Gainesville, Florida 32610, United States.
authors.forename: Anamika
authors.initials: A
authors.lastname: Singh
authors.forename: Marvin L
authors.initials: ML
authors.lastname: Dirain
authors.forename: Andrzej
authors.initials: A
authors.lastname: Wilczynski
authors.forename: Chi
authors.initials: C
authors.lastname: Chen
authors.forename: Blake A
authors.initials: BA
authors.lastname: Gosnell
authors.forename: Allen S
authors.initials: AS
authors.lastname: Levine
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Edison
authors.forename: Carrie
authors.initials: C
authors.lastname: Haskell-Luevano
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical; D004435:Eating / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D018377:Neurotransmitter Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000494:pharmacology*; D066244:Proton Magnetic Resonance Spectroscopy; D051381:Rats; D017207:Rats, Sprague-Dawley; D044104:Receptor, Melanocortin, Type 3 / Q000819:agonists / Q000378:metabolism; D044105:Receptor, Melanocortin, Type 4 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000521:alpha-MSH / Q000378:metabolism
chemical_list: C000599368:AST3-88 peptide; C574273:MC4R protein, mouse; C470053:Mc3r protein, mouse; D018377:Neurotransmitter Agents; D010456:Peptides, Cyclic; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D000521:alpha-MSH
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