Arg-Phe-Phe d-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles

Abstract

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized β-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

Peptoid NPhe4 in AGRP-Based c[Pro1-Arg2-Phe3-Phe4-Xxx5-Ala6-Phe7-DPro8] Scaffolds Maintain Mouse MC4R Antagonist Potency

“…Substitution of NPhe at the Phe 4 resulted in the most potent MC4R antagonist assayed with Dap at position 5 ( 4 ). The equivalent NPhe 4 substitution when Asn was at position 5 ( 11 ) was the only Asn 5 peptide-peptoid macrocycle to possess antagonist potency at the MC4R and was equipotent to previous reports of the amino acid only form (reported pA 2 values at the MC4R range from 7.7 to 8.2). ,, A previous report inverting the stereochemistry at this position in the macrocyclic scaffold, resulting in DPhe 4 , decreased antagonist potency greater than 10-fold . It therefore appears that the loss of stereochemistry alone does not result in the increased potency observed for the NPhe substitutions.…”

“…Similar to prior reports, inverse agonist activity was observed for select ligands at the MC5R. ,, Due to the increased MC4R antagonist potency, three compounds were examined for antagonist potency using a Schild experimental design at the MC5R, the parent 1 and the NPhe 4 containing 4 and 11 . The parent 1 was previously reported to possess nanomolar MC5R antagonist potency (pA 2 = 7.3), similar to the value observed in the present study (pA 2 = 7.5).…”

“…The equivalent Dap 5 macrocycles with NPhe 3 ( 3 ) and NAla 6 ( 5 ) resulted in partial MC1R agonism (80% the maximal NDP-MSH signal) or no agonist activity at up to 100 μM concentrations, respectively. A D-amino acid scan of the Arg-Phe-Phe tripeptide sequence did not result in a similar agonist activity pattern at the MC1R . This observed MC1R agonism may suggest a peptoid substitution pattern that may be exploited in generated selective, potent MC1R agonist ligands.…”

“…25,26,44 A previous report inverting the stereochemistry at this position in the macrocyclic scaffold, resulting in DPhe 4 , decreased antagonist potency greater than 10-fold. 41 It therefore appears that the loss of stereochemistry alone does not result in the increased potency observed for the NPhe substitutions. Antagonist potency at the MC4R is also maintained when a hPhe (the Phe side chain is extended by one methylene unit) is inserted into position 4.…”

“…Peptides were synthesized using standard Fmoc techniques, , as previously described. ,,, Due to incomplete coupling following the addition of the Fmoc-NPhe residue at select positions, the synthetic procedure was modified as noted in the experimental section. Peptides were purified to greater than 95% purity in two diverse solvent systems (Table ), and the correct molecular weight was determined by ESI-MS or MALDI-MS (University of Minnesota Mass Spectrometry Lab).…”