Discovery of Melanocortin Ligands via a Double Simultaneous Substitution Strategy Based on the Ac-His-dPhe-Arg-Trp-NH2 Template

Abstract

The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His¹-dPhe²-Arg³-Trp⁴-NH₂ tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1-5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His¹-dPhe²-Arg³-Trp⁴-NH₂ tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4, as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at positions 1 and 2, (C) double simultaneous substitution at positions 1 and 4, and (D) double simultaneous substitution at positions 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency or selectivity or converting agonists into antagonists and vice versa.

Keywords: MC5R; SAR; alpha-MSH; melanotropin; selective ligands.

Figure 1.

(A) Illustration of the truncation strategy approach. The concept is based on the shortening of the full-length peptide in order to obtain the minimal sequence required to elicit a pharmacological response at the target receptors. The truncation can be performed in the C-terminus to N-terminus direction (as shown) or vice versa. (B) Illustration of the positional scanning approach that is based on the altering of one amino acid position at the time while the remaining positions are kept constant. (C) Illustration of alanine scanning which is a special case of positional scanning in which the amino acid used for positional scanning is alanine.

Figure 2.

Illustration of the double simultaneous substitution technique. The strategy applied in this approach relies on the two concurrent substitutions while the other positions are kept constant. Various amino acids are simultaneously introduced at positions 1, 2, or 4 as well as the N-terminal capping position. The following combinations are performed: (A) capping group position (Ac) together with position 1, 2, or 4, (B) position 1 and 2, (C) position 1 and 4, and (D) position 2 and 4.

Figure 3.

Chemical structures of amino acids used as diversity elements in the double simultaneous substitution approach.

Figure 4.

Illustration of the double simultaneous substitution approach denoting various amino acids used at the specific positions in the Ac-His-DPhe-Arg-Trp-NH₂ tetrapeptide template.

Figure 5.

Graphical illustration of ligands that possess no antagonist activity at the melanocortin receptors. All compounds presented this figure possess full agonist profile at melanocortin receptors examined in this study.

Figure 6.

Graphical illustration of ligands that possess no antagonist activity at the melanocortin receptors. All compounds presented this figure possess full agonist profile at mMC1R, mMC4R, and mMC5R.

Figure 7.

Illustration of the competitive antagonism of peptide 12 at the mMC3R as assessed by Schild analysis. Note that there is no agonist activity by compound 12 alone at concentrations up to 100 μM.

Figure 8.

Pharmacological profile of the peptide 23 that is a selective nanomolar agonist for the mMC4R, as opposed to its minimal agonist activity at the mMC3R.