Demonstration of a common DPhe⁷ to DNal(2’)⁷ peptide ligand antagonist switch for the melanocortin-3 and melanocortin-4 receptors identifies systematic mischaracterization of the pharmacological properties of melanocortin peptides

Abstract

Melanocortin peptides containing a D-naphthylalanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative PCR. While the MC1R cell line correctly expressed only the hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. Demonstration that a D-naphthylalanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in antagonism of the MC3R and MC4R, then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

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“…The above data are consistent with previously reported evidence that MC1R and MC5R have relatively few antagonists in MC. , This is unlike MC3R and MC4R, where sequential studies of the MC pharmacophore led to the use of DNal(2′) substitution for the histidyl residue in the MC pharmacophore, producing potent receptor antagonism in many MC analogs. − Accordingly, TCMCB07, which contains DNal(2′), is an MC3R and MC4R antagonist but is an agonist for MC1R and MC5R (Figures , , and , and Tables , , and ).…”

“…Most MC peptides with a DNal(2′) residue are antagonists or have weak partial agonism for hMC3R/hMC4R. − , This phenomenon also applies to TCMCB07, acting as antagonist on MC3R and MC4R of humans, rats, and dogs (Figures and , and Tables and ). Additionally, pA2 is the estimated equilibrium dissociation constant for the antagonist, which is the dose of antagonist that requires a 2-fold increase in agonist concentration .…”

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“…16,17 Similar to the previously reported MC4-R:SHU9119 (PDB: 6W25) complex structure, 16 the newly solved receptor:PG-934 and SBL-MC-31 costructures showed a robust and Ca 2+ -suggestive electron density. Equally to the receptor:SHU9119 complex, Ca 2+ was coordinated by the receptor acidic E100 2.60 and D122 3.25 side chain carboxyl oxygens and both D126 S4), highlighting the stabilizing effect of many macrocyclic rings on Ca 2+ binding, regardless of their agonist or antagonist activity on MC4-R. For instance, SHU9119 has partial agonist activity on MC1-R, 46 but the same coordination network for this ligand was still described in the MC1-R:SHU9119 (PDB: 7F4I) and MC1-R:Nle 4 -DPhe 7 α-MSH (PDB: 7F4F) complexes 14 and even linear or cyclic peptides with MC2-R, (PDB: 8GY7) MC3-R (PDB: 8IOC), and MC5-R (PDBs: 8INR and 8IOD). 15,20 To further elucidate the role of Ca 2+ on ligand potencies and receptor stability, we assessed the effect of MC4-R E100 2.60 , D122 3.25 , and D126 3.29 two MC4-R agonists to overcome the direct impact of Ca 2+ on agonist responses.…”

“…These responses mimicked SHU9119, with full antagonist activity at MC4-R, low partial agonist, almost full antagonist activity at MC3-R, mid partial agonist activity at MC1-R, and full agonist activity at MC5-R (Tables 3 and 4 and Figure S8). 46 Although none of the peptides showed significantly increased potency for MC4-R, a selected subset showed increased IC 50 values (i.e., decreased potency) for the inhibition of α-MSH elicited cAMP accumulation (SBL-MC-71, SBL-MC-72, SBL-MC-73, SBL-MC-75, and SBL-MC-76, Figure 5A and Table 3). Notably, a peptide subset that incorporated a less flexible S-triazole substitution at the Pro 6 pyrrole ring position four rather than the aliphatic chains present in the other peptides (Figure 4), with the longest side chain containing peptides (SBL-MC-72 and SBL-MC-76) causing the most significant hindrance in potency among the PG-934-derived series (Figure 5A).…”

“…As previously described, cell line identity was periodically verified by qPCR and melanocortin receptor subtype-specific oligonucleotides. 46 Twenty-four hours before the cAMP response recordings, the cells for each receptor subtype were plated in poly-D-lysine-coated clearbottom black wall 384-well plates (Corning Inc. Corning, NJ) at a density of 20,000 cells per well in complete growth medium. Two hours before the start of the experiment, the growth medium was replaced with CO 2 Independent Medium (Thermo Fisher Scientific), containing 4% D-luciferin (Promega).…”