Elsevier

Peptides

Volume 21, Issue 2, February 2000, Pages 239-243
Peptides

Regular Papers
New highly specific agonistic peptides for human melanocortin MC1 receptor☆

https://doi.org/10.1016/S0196-9781(99)00207-7Get rights and content

Abstract

A peptide with very high specificity for the human melanocortin MC1 receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC1 receptor. Both these peptides showed strong agonistic activity at the MC1 receptor. The MS05 was the most MC1 receptor selective as it showed virtually no binding affinity for the MC4 and MC5 receptors and only micromolar affinity for the MC3 receptor. The selectivity and potency of the new peptides make them potent tools for studies of MC1 receptors, as well as novel potential candidate drugs for the treatment of inflammatory conditions.

Introduction

The five presently known melanocortin receptors (MC1–5) belong to the superfamily of G protein coupled receptors and are expressed in many different tissues. Their natural ligands, the melanocortin peptides, were recognized a long time ago for their effects on pigment formation and granulae dispersion in melanocytes and melanophores, and for their ability to regulate steroid production in the adrenal gland. However, many early and recent studies recognize that the melanocortins also display other effects, including the regulation of the immune system [5], [11], [24]. The melanocortins have therefore been recognized as a family of neuroimmunomodulatory peptides [24].

The first MC receptors cloned were the MC1 receptor of the melanocytes [3], [12], and the MC2 (ACTH) receptor of the adrenal gland [12]. Soon thereafter followed the cloning of three other melanocortin receptor subtypes termed MC3–5 [4], [6], [7], [8]. The mapping of the functions and distributions of the MC receptors is in progress, the most prominent recent results being the recognition of the MC1 receptor as a potential modulator of the immune system and the MC4 receptor as a key receptor in control of feeding homeostasis (see Ref. [25]).

All of the melanocortin receptors, except the MC2, bind the 13 amino acid-long α-MSH peptide, as well as structurally related α-MSH peptide analogs [16], [17]. A few synthetic melanocortin receptor selective ligands have emerged. Adan et al. [1] reported that some ACTH [4], [5], [6], [7], [8], [9], [10] analogs showed antagonistic properties on MC4 receptors. More recently, some cyclic peptides possessing high affinity and antagonistic properties on MC4 receptors were described [9], [10], [21]. With the help of phage display and by using insect cell expressing high densities of a modified MC1 receptor [18] as a selection tool, we recently identified a peptide that binds to the MC1 receptor with very high selectivity [23]. With this peptide as a lead we describe the synthesis of three novel peptides that show agonistic activity on MC1 receptors. Of these, the MS05 and MS09 peptides were found to bind with sub-nanomolar affinity for the MC1 receptor. The MS05 peptide was the most selective and it shows hitherto unrivaled selectivity for the MC1 receptor.

Section snippets

Materials and methods

Chemicals were obtained from Sigma (St. Louis, MO, USA), radionucleides from Amersham (Arlington Heights, IL, USA), and α-MSH and [Nle4, D-Phe7]α-MSH (NDP-MSH) from Bachem (Torrance, CA, USA). All consumables and media for cell culture were obtained from LifeTechnologies (Rockville, MD, USA).

Peptides were synthesized on solid phase by using continuous flow Fmoc chemistry in a Pioneer Peptide Synthesis System (PerSeptive Biosystems, Cambridge, MA, USA). Raw products were purified by reverse

Results

Three novel peptides were designed by combining the amino acid sequences of the α-MSH, NDP-MSH, and the MS04 peptides, the latter obtained in an earlier study by selection of a phage display library using MC1 receptor expressing insect cells as a selection tool [23]. The amino acid sequences of the peptides are shown in Table 1. Thus, the MS05 peptide was obtained by substituting the three C-terminal amino acids of MS04 with the three C-terminal amino acids of α-MSH. The MS06 peptide was

Discussion

We have here derived a series of peptides by using the MS04 peptide as a lead. The MS04 we originally obtained from a phage display library by using MC1 receptor-expressing cells a selection tool [23]. MS04 is highly specific for the MC1 receptor, but it shows only a moderate affinity for MC1 receptor compared to many other known peptide ligands. The MS04 sequence also contained a cysteine residue, which renders both its synthesis and handling difficult. Moreover, MS04 is a partial agonist [23]

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☆ This work was supported by a grant from the Swedish Medical Research Council (04X05957).

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