Structure−Activity Relationships of the Melanocortin Tetrapeptide Ac-His-DPhe-Arg-Trp-NH₂ at the Mouse Melanocortin Receptors. 1. Modifications at the His Position

Abstract

The melanocortin pathway is an important participant in obesity and energy homeostasis. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). The melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp", and it has been well documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library based on the template Ac-His-DPhe-Arg-Trp-NH(2), consisting of 17 members that have been modified at the His(6) position (alpha-MSH numbering) and pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. These studies provide further experimental evidence that the His(6) position can determine MC4R versus MC3R agonist selectivity and that chemically nonreactive side chains may be substituted for the imidazole ring (generally needs to be side chain protected in synthetic schemes) in the design of MC4R-selective, small-molecule, non-peptide agonists. Specifically, the tetrapeptide containing the amino-2-naphthylcarboxylic acid (Anc) amino acid at the His position resulted in a potent agonist at the mMC4R (EC(50) = 21 nM), was a weak mMC3R micromolar antagonist (pA(2) = 5.6, K(i) = 2.5 microM), and possessed >4700-fold agonist selectivity for the MC4R versus the MC3R. Substitution of the His(6) amino acid in the tetrapeptide template by the Phe, Anc, 3-(2-thienyl)alanine (2Thi), and 3-(4-pyridinyl)alanine (4-Pal) resulted in equipotency or only up to a 7-fold decrease in potency, compared to the His(6)-containing tetrapeptide at the mMC4R, demonstrating that these amino acid side chains may be substituted for the imidazole in the design of MC4R-selective non-peptide molecules.

Exposure of MC4R to agonist in the endoplasmic reticulum stabilizes an active conformation of the receptor that does not desensitize

“…2, lanes 7 and 8). This result is consistent with the expected inability of α-MSHdelta to act as an agonist (40) and with the observation that ER-α-MSH-delta does not appear to bind to HA-MC4R-GFP (Fig. 1).…”

“…2, lanes 6 and 2). This result is consistent with the observation that mutation of His 6 to Trp 6 within α-MSH reduces potency toward MC4R (40). These experiments indicate that the expression of intracellular α-MSH leads to cAMP signaling that is constant and that, when the tetrapeptide motif His 6 -Phe 7 -Arg 8 -Trp 9 of α-MSH is intact, has the same amplitude as the signaling induced by extracellular α-MSH.…”

“…Posttranslational processing of POMC generates α-, β-, and γ-(γ-MSH) melanocyte-stimulating hormones and ACTH. These hormones are agonists of MC4R and of other melanocortin receptors (40,41). To target α-MSH to the ER, we generated a construct in which the signal sequence of POMC directing the hormone precursor to the ER was retained and was followed directly by the sequence of α-MSH with a Myctag at the C terminus (ER-α-MSH).…”

“…To target α-MSH to the ER, we generated a construct in which the signal sequence of POMC directing the hormone precursor to the ER was retained and was followed directly by the sequence of α-MSH with a Myctag at the C terminus (ER-α-MSH). The tetrapeptide motif His 6 -Phe 7 -Arg 8 -Trp 9 of α-MSH is essential for stimulation of MC4R (40,(42)(43)(44), and mutation of His 6 to Trp 6 within this tetrapeptide reduces potency toward MC4R (40). We generated constructs with α-MSH lacking the tetrapeptide motif (ER-α-MSH-delta) and with His 6 mutated to Trp 6 (ER-α-MSH-Trp) (Fig.…”

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

“…␣-MSH shares the first 13 amino acids with ACTH, and they share the core sequence His 6 -Phe 7 -Arg 8 -Trp 9 . This region was identified as important for ligand binding and activities at hMC1R, hMC2R, hMC3R, hMC4R, and hMC5R (27)(28)(29)(30)(31)(32)(33)(34). Our previous results indicate that [Nle 4 ]ACTH(1-39) (with norleucine substituted for methionine at position 4) is equipotent to ACTH(1-39) in ligand binding and signaling (35).…”