A Macrocyclic Agouti-Related Protein/[Nle⁴,DPhe⁷]α-Melanocyte Stimulating Hormone Chimeric Scaffold Produces Subnanomolar Melanocortin Receptor Ligands

Abstract

The melanocortin system consists of five receptor subtypes, endogenous agonists, and naturally occurring antagonists. These receptors and ligands have been implicated in numerous biological pathways including processes linked to obesity and food intake. Herein, a truncation structure-activity relationship study of chimeric agouti-related protein (AGRP)/[Nle4, DPhe7]α-Melanocyte Stimulating Hormone (NDP-MSH) ligands is reported. The tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp replaced the Arg-Phe-Phe sequence in the AGRP active loop derivative c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was the native Asn of AGRP or a diaminopropionic (Dap) acid residue previously shown to increase antagonist potency at the mMC4R. The Phe, Ala, and Dap/Asn residues were successively removed to generate a 14-member library that was assayed for agonist activity at the mouse MC1R, MC3R, MC4R, and MC5R. Two compounds possessed nanomolar agonist potency at the mMC4R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro], and may be further developed to generate novel melanocortin probes and ligands for understanding and treating obesity.

Structure–Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor

“…54 To further investigate this scaffold, a series of truncated macrocycles were synthesized containing either the Asn or Dap residues and replacing the Arg-Phe-Phe tripeptide antagonist sequence with the melanocortin agonist tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp sequences. 55 This study examined if the AGRP DPro-Pro loop scaffold was amendable to substitutions that may result in melanocortin agonist activity as weight-management therapeutic leads. Two macrocycles were identified that possessed nanomolar agonist potencies at the MC4R, nanomolar to sub-nanomolar potencies at the MC1R and MC5R, and 30–40 nanomolar potencies at the MC3R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro].…”

“…Two macrocycles were identified that possessed nanomolar agonist potencies at the MC4R, nanomolar to sub-nanomolar potencies at the MC1R and MC5R, and 30–40 nanomolar potencies at the MC3R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro]. 55 …”

Comparative Intracerebroventricular and Intrathecal Administration of a Nanomolar Macrocyclic Melanocortin Receptor Agonist MDE6-5-2c (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro]) Decreases Food Intake in Mice

“…Food intake results are consistent with the in vitro pharmacology of MDE6-5-2c functioning as a nonselective MC3R and MC4R agonist. 33 It was postulated that a dose-dependent decrease in cumulative food intake would result with 2, 5, and 10 nmol MDE6-5-2c concentrations via ICV administration. A dose-dependent decrease in cumulative food intake was not observed, however, when MDE6-5-2c was administered IT.…”

“…An antagonist AGRP based macrocyclic chemotype scaffold was modified and converted to the potent nonselective MC3R/MC4R agonist MDE6-5-2c. 33 Herein, intrathecal (IT) and intracerebroventricular (ICV) routes of administration of MDE6-5-2c were used to compare differences in energy homeostasis profiles when administered directly into the brain (ICV) or spinal cord (IT). Both ICV and IT administration of 5 nmol MDE6-5-2c decreased food intake as compared to vehicle control treated mice.…”

“…It has previously been reported that cyclizing the active loop of AGRP head-to-tail through a DPro-Pro motif, yielding the macrocycle c­[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro], resulted in a nanomolar MC4R antagonist ligand . Further structure activity–relationship (SAR) studies demonstrated that replacing the Asn with a diaminopropionic acid (Dap) residue increased MC4R antagonist potency, resulting in an equipotent MC4R antagonist compared to AGRP. − To generate agonist ligands, the Arg-Phe-Phe tripeptide sequence from the antagonist macrocyclic scaffold was replaced with the His-DPhe-Arg-Trp melanocortin agonist sequence . Truncation studies established that removing the Phe from the scaffold, generating the macrocyclic octapeptide c­[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro] (MDE6-5-2c), resulted in an agonist ligand possessing EC 50 potencies of 1, 40, 2, and 0.3 nM at the MC1R, MC3R, MC4R, and MC5R, respectively .…”