d-Amino Acid Scan of γ-Melanocyte-Stimulating Hormone:  Importance of Trp⁸ on Human MC3 Receptor Selectivity

Abstract

In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.

A role for MC3R in modulating lung inflammation

“…We have done so by using a pharmacological, genetic and molecular approach utilising the selective MC3R ligand [D-TRP 8 ]-γ-MSH, [24,25], MC3R deficient mice [28,31] and the recessive yellow e/e (bearing a nonfunctional MC1R) mouse [14]. This study has focused on the MC1 and 3R given the wealth of data highlighting that both these receptors are involved in modulating the host inflammatory response [10] other MCR were not evaluated in this study due to the fact that they haven't been implicated in modulating peripheral inflammation.…”

“…Following electrophorysis in a 10% polyacrylamide gel, membranes were blocked overnight in blocking solution containing 5% non-fat milk solution in Tris buffer saline containing 0.1% Tween-20 followed by anti-MC3R (1:2000) [22] synthesised by Dr Paolo Grieco as previously described [24,25], peptides were stored at -20°C prior to use, and dissolved in sterile PBS (pH 7.4).…”

Agouti-Related Peptide and MC3/4 Receptor Agonists Both Inhibit Excitatory Hypothalamic Ventromedial Nucleus Neurons

“…9 A, C,D). D-Trp8-␥-MSH was used as a selective agonist of MC3R given its 40-to 200-fold selectivity over MC4R and MC5R (Grieco et al, 2000;Cowley et al, 2001). In all cells tested, D-Trp8-␥-MSH (10 nM) (1 min) also showed a consistent inhibition of GFP-VMH neurons (Fig.…”

Design, Synthesis and Biological Evaluation of Ligands Selective for the Melanocortin-3 Receptor

“…[e.g. 41,43]. Once the structure activity relationships (SAR) of the peptide ligand have been elucidated, the conformation-activity relationships have to be examined.…”

“…Biological screening demonstrated that γ-MSH was selective for the hMC3R by up to 100 fold, compared to α-MSH [49]. Grieco et al used a D-amino acid scan of the γ-MSH sequence to obtain the first highly selective agonist for the hMC3R (H-Tyr-Val-Met-Gly-His-Phe-Arg-D-Trp-Asp-Arg-PheGly-OH; EC 50 = 0.33 nM; hMC4R/hMC3R = 300, hMC5R/hMC3R = 250) [43]. Furthermore, replacement of the oxidizable Met 3 residue with Nle increased the stability of the peptide [52], which is being widely used as a ligand for the hMC3R in a variety of ongoing pharmacological studies and in animal model studies related to the hMC3R.…”