Original article
Design of new small cyclic melanocortin receptor-binding peptides using molecular modelling: Role of the His residue in the melanocortin peptide core

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Abstract

The conserved core of melanocyte stimulating hormones (MSH), His-Phe-Arg-Trp, was probed by comparing a cyclic pentapeptide containing His-dPhe-Arg-Trp, with three structurally similar cyclic peptides, that lacked the His residue. All three peptides bound to the MC1, MC3, MC4 and MC5 receptors with similar affinities. Molecular modelling indicated that the 3D structure of the dPhe-Arg-Trp of all three peptides were closely similar. The data indicate that the His residue of the small rigid cyclic MSH core peptides does not participate in binding with the melanocortin receptors.

Introduction

Melanocortin receptors are members of the G-protein coupled receptor superfamilly. There are five subtypes of the melanocortin receptors (MC1–MC5), all which were cloned a few years ago [1], [2], [3], [4], [5]. The MC1 receptor has been found in melanocytes where it controls skin pigmentation, as well as in the periaqueductal grey area of the brain [6]. More recent data indicated that the MC1 receptor is also present in cells of the immune system [7]. MC3, MC4 and MC5 receptors are localised in the central nervous system; the MC3 and MC5 receptors also in periphery [1], [3], [4], [8]. Recent studies indicate that the MC4 receptor is involved in control of feeding behaviour [9] and the MC5 receptor in control of exocrine gland function [10]. The MC2 receptor has distinct structural and binding properties [11] and it is localised in the adrenal cortex [12].

Melanocyte stimulating hormones (MSH) are natural ligands for four of the melanocortin receptor subtypes, namely the MC1, MC3, MC4 and MC5 receptors. These hormones are peptides of more than 10 amino acids in length that bind with higher affinity to the MC1 receptor, than to the MC3, MC4 and MC5 receptors [13], [14], and which share a common core sequence motif: HFRW [15].

A number of artificial linear and cyclic MSH peptide analogues have been synthesised, most of which contain the core sequence. Most of these substances show quite similar selectivity profiles to the natural hormones [15]. Moreover, it was shown that exchange of the l-Phe residue in the core sequence to d-Phe generally increases the affinities for the melanocortin receptors [16].

An alanine scan of the α-MSH peptide indicated that the core sequence plays a key role in the binding of melanocortin hormones to their receptors, although the available data suggests that the importance of the His residue may be lower, compared to the other three core amino acids [17].

However, it has been shown that the minimal active α-MSH peptide fragment on lizard skin assay was the Ac-His-Phe-Arg-Trp-NH2 peptide, which contains the His residue [18]. In a later study on small linear tripeptides containing d-amino acids in different positions, it was found that none of these peptides were able to bind to MC1, MC3 and MC5 receptors, while two peptides (Ac-dPhe-Arg-Trp-NH2 and Ac-dPhe-Arg-dTrp-NH2) bound to the MC4 receptor [19].

In our attempts to understand the pharmacophore of the melanocortin receptors, we recently made a 3D model of the MC1 receptor using homology modelling, based on the rhodopsin footprint [20]. In these attempts, we also designed a small cyclic MSH peptide analogue that contained the core sequence with l-Phe exchanged to d-Phe. The smallest of these peptides was a pentapetide, cHdFRWG (1), which was found to bind with substantial affinity to the melanocortin receptors, the highest affinity being shown for the MC1 receptor [21]. The peptide (1), which just contains the core sequence and glycine, was subsequently successfully docked into our MC1 receptor model [18]. However, these docking studies indicated that only three of the core residues, namely Phe, Arg and Trp, were strongly involved in binding of the cHdFRWG (1) peptide to the receptor, while the interactions with the His residue appeared to be minor.

The present study was devoted to test the hypothesis that the His residue of the cyclic core peptides does not have a major role for the melanocortin receptor binding. For the purpose of these studies we designed three new cyclic peptides; one being a cHdFRWG analogue in which histidine was replaced by asparagine: cNdFRWG (2), and the other two being the cyclic peptides c(dFRWE)OH (3) and c(dFRWE)NH2 (4) which contain the d-Phe, Arg and Trp core residues, and Glu, which side chains were cyclised with the N-terminal of the d-Phe residue (see figure 1). In support of our hypothesis, we report here that these histidine-lacking peptides bind with equal or higher affinity to the melanocortin receptors, compared with cHdFRWG (1).

Section snippets

Binding

The binding of (2), (3) and (4) was tested on intact CV-1 origin, SV40 (COS-1) cells expressing the human MC1, MC3, MC4 and MC5 receptors using competitive receptor-binding assays. The resulting Ki values, obtained from competition tests of these peptides, are summarised in table I along with the corresponding data for the cHdFRWG (1) peptide taken from our earlier study [21]. Competition curves for (2), (3) and (4) are shown in figure 2. The data show that all four peptides share similar

Discussion

Our data show that the cNdFRWG (2), c(dFRWE)OH (3) and c(dFRWE)NH2 (4) peptides possess similar or even improved binding affinities for the melanocortin receptors compared to the cHdFRWG (1) peptide. Thus, the loss of the His residue does not result in any loss in binding affinities compared to the cHdFRWG (1) peptide; instead, a clear increase in binding affinity is seen for the cNdFRWG (2) peptide for the MC3, MC4 and MC5 receptors, while for the c(dFRWE)NH2 (4) peptide, an increase in

Synthesis and characterisation of cyclic peptides

Reagents were purchased from Aldrich, Fluka and Applied Biosystems. Solid phase synthesis of peptides was made on a Pioneer peptide synthesis system (Applied Biosystems), as is detailed further below. LC/MS was performed on a Perkin Elmer instrument PE SCIEX API 150EX with Turboionspray Ion Source and Vydac 214 MS 52 C4 MASS SPEC 2.1×250 mm high performance liquid chromatography (HPLC) column using a gradient formed from water and acetonitrile with 0.02% trifluoroacetic acid (TFA) additive

Acknowledgements

Support was obtained from the Swedish MRC (04X-05957).

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