Synthesis and Pharmacological Characterization of Adrenocorticotropic Hormone (ACTH 1–24) and C-Terminal Truncated Analogues Identifies the Minimal ACTH N-Terminal Fragment Required for Melanocorton-2 Receptor Activation

Purpose: The melanocortin system has been implicated in regulating various physiological pathways including skin and hair pigmentation, energy homeostasis, food intake, steroidogenesis, and exocrine gland regulation. Five melanocortin receptors (MCRs) have been identified to date that are activated by agonist peptide ligands derived from the proopiomelanocortin (POMC) prohormone. While the MC1R, MC3R, MC4R, and MC5R can be activated by several of these ligands, the MC2R is unique in that it is only activated by the adrenocorticotropic hormone (ACTH). The aim of the present study was to identify the minimal N-terminal fragment of ACTH that can fully stimulate the MC2R. Methods: A series of 12 peptides, representing C- to N-terminal truncated analogs from the starting ACTH (1–24) ligand, were synthesized and pharmacologically characterized in parallel at the five melanocortin receptors. Results: While truncation had minimal effects (≤6-fold) at the MC1R, MC3R, MC4R, and MC5R, the basic tetrapeptide Lys-Lys-Arg-Arg sequence corresponding to residues 15–18 of ACTH was identified to be critical for agonist potency at the MC2R. Sequential removal of these residues decreased potency 8-, 114-, 1000-, and >6500-fold relative to the ACTH(1–24) ligand, with the minimal sequence stimulating the MC2R being ACTH(1–15) EC₅₀ = 1450 nM). Conclusion: These results correlate to the cryo-EM structure reported in 2023 of ACTH-MC2R-MRAP1 which showed these basic ACTH residues make several interactions with MRAP1.