4 December 2019
One of the first lines of cutaneous defense against photoaging is (a) the synthesis of melanin and (b) the initiation of an oxidative stress response to protect skin against the harmful effects of solar radiation. Safe and selective means to stimulate epidermal pigmentation associated with oxidative stress defense are; however, scarce. Activation of the melanocortin-1 receptor (MC1R) on epidermal melanocytes represents a key step in cutaneous pigmentation initiation and, additionally, it regulates cellular defense mechanisms like oxidative stress and DNA-repair. Thus, making the activation of MC1R an attractive strategy for modulating skin pigmentation and oxidative stress. In this context, we designed and synthesized pentapeptides that act as MC1R agonists. These peptides bound, with high potency, to MC1R and activated cAMP synthesis in CHO cells expressing human MC1R. Using one lead pentapeptide, we could show that this activation of MC1R was specific as testing the activation of other G-protein coupled receptors, including the MC-receptor family, was negative. In vitro efficacy on mouse melanoma cells showed similar potency as for the synthetic MC1R agonist alpha-melanocyte stimulating hormone (NDP-alpha-MSH). Moreover, we could reproduce this activity in human skin tissue culture. The lead pentapeptide was able to induce ex-vivo protein expression of key melanogenesis markers melanocyte inducing transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP-1). Concerning oxidative stress response, we found that the pentapeptide enhanced the activation of Nrf2 after UVA-irradiation. Our results make this pentapeptide an ideal candidate as a skin pigmentation enhancer that mimics alpha-MSH and may also have anti-photoaging effects on the skin.
Melanocortin-1 receptor (MC1R) is a seven-transmembrane Gs-protein coupled receptor (GPCR) present on the surface of melanocytes [1,2]. MC1R belongs to a family of five melanocortin receptors (MCRs) (MC1R, MC2R, MC3R, MC4R, and MC5R) [3,4,5,6,7,8] which are involved in key biological functions including pigmentation, oxidative stress response, DNA-damage repair, immune responses, feeding behavior, energy homeostasis, sexual function, and sebaceous gland secretion [9,10,11,12]. The natural agonist ligands for the MCRs are derived from proopiomelanocortin (POMC), a polypeptide of 241 amino acids. Four endogenous MCR agonist peptides have been identified including α, β, and γ-melanocyte stimulating hormones (MSHs) and the adrenocorticotropic hormone (ACTH) [13]. These are generated from POMC by proteolytic cleavage by prohormone convertase 1/3 and prohormone convertase 2 [9,10]. alpha-MSH, a tridecaptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp- Gly-Lys-Pro-Val-NH 2, binds preferentially to MC1R and is the most important MSH in stimulating melanogenesis and thus cutaneous pigmentation.
Melanin in the skin serves mainly to protect cellular DNA from ultraviolet radiation (UVR)-induced DNA damage [14]. It has a broad absorption spectrum ranging from UV into visible light up to 700 nm [15]. It typically forms supranuclear caps absorbing about 50%–75% of UVR [16] and thus acts as a natural shield against DNA-damage and photoaging. DNA-damage in both keratinocytes, as well as melanocytes, can lead to various cutaneous abnormalities, like actinic keratosis, and in more severe cases, basal as well as squamous cell carcinoma and melanoma [17]. Upon DNA-damage evoked by UVR, alpha-MSH is synthesized and secreted by keratinocytes. It binds to MC1R expressed on the surface of melanocytes to activate the receptor [2]. MC1R is constitutively expressed in human epidermis, but it can be further induced by UVR [18,19,20]. Activation of MC1R sets in motion a signaling cascade through cyclic adenosine monophosphate (cAMP) which leads to the transcription of the genes for melanogenesis. First, the key transcription factor melanocyte inducing transcription factor (MITF) is induced. MITF initiates the transcription of various down-stream targets like tyrosinase (TYR), the rate-limiting enzyme in melanogenesis, and tyrosinase-related protein 1 (TYRP-1), which converts 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into eumelanin [17]. Common pigmentation disorders include vitiligo [21], solar lentigo [22], and age spots [23], which are not only of dermatological concern but also receive ample attention from aestheticians and the cosmetic industry. They have in common that they appear as uneven skin tone. Uneven skin tone is of aesthetic concern in most societies and an even skin tone, be it dark or bright, is preferred [24,25]. The color of the skin is perceived by the human eye as light scattered by chromophores in the skin like, for example, mainly melanin, but also hemoglobin, oxy-hemoglobin, bilirubin, and carotenoids [26]. Thus, a change in one or more of these chromophores affects skin color.
Besides its role in melanogenesis initiation, the alpha-MSH-MC1R system has anti-oxidative properties. It was shown that it can induce nuclear factor erythroid 2 related factor 2 (Nrf2) [27], a key transcription factor in the cellular defense against oxidative stress evoked by UVR [28]. Nrf2 dissociates from its inhibitor Keap1 upon phosphorylation on serine 40, which leads to antioxidant response element mediated transcription [29] of antioxidant and phase II detoxifying enzymes like heme oxygenase-1 [30]. With respect to DNA-damage repair, activation of MC1R by alpha-MSH is positively involved in nucleotide-excision repair [31], and it ameliorated oxidative stress-induced formation of 7,8-dihydro-8-oxyguanine (8-oxodG), a major form of oxidative DNA damage [12]. Hence, the alpha-MSH-MC1R pathway serves as a cellular multiple-line of defense system against the damaging effects of UVR.
Due to the above-mentioned signaling cascades, MC1R is also a target for anti-aging strategies. It is well established that UVR, the main contributor to photoaging, induces oxidative stress, inflammation, and activation of matrix metalloproteases (MMPs) [32,33], which degrade extracellular matrix (ECM) components such as collagens and elastic fibers [34]. This leads to visible signs of aging like wrinkles and skin sagging [35,36,37]. It was found that darkly pigmented skin shows around 10 years delayed signs of photoaging, possibly due to the photo-protective effects of melanin [38,39,40]. In addition, melanocytes are part of a sophisticated and complex skin neuroendocrine network [41]. As such, they secrete neuroendocrine signals essential for cutaneous homeostasis [41,42]. Aging leads to the decline of biochemical and physiological mechanisms also with respect to the skin neuroendocrine system [43]. As such, it was shown that aging affected the expression of MC1R as well as POMC and its derived MC1R-agonist peptides [43,44,45]. Specifically, a marked decrease in MC1R expression was shown in the skin from 60-year-old donors compared to 30-year-old donors, while expression of POMC increased with age in basal keratinocytes but was unaffected in total epidermis [45]. Furthermore, genome-wide association studies found multiple single nucleotide polymorphisms (SNPs) within the MC1R gene and other pigmentation associated genes contributing to perceived facial age [46,47].
The alpha-MSH analog NDP-alpha-MSH [Nle 4, D-Phe 7], also known as Melanotan-1 or afamelanotide (MT-I), is a synthetic peptide that induces skin pigmentation [48] and has been approved in Europe for treating erythropoietic protoporphyria (EPP), a skin disease involving phototoxicity which can be ameliorated by inducing skin pigmentation. MT-1 is a universal MCR agonist which can lead to unexpected side effects such as headache and nausea [49].
The search for a smaller more selective and safer peptide sequence that mimics alpha-MSH has been the objective of many research groups [31,50,51]. In addition, the search for molecules with UV-independent pro-pigmenting activity cumulated in the finding of a small salt-inducible kinase inhibitor with melanogenic activity in mice and human skin explants [52].
In this study, we designed and synthesized a novel alpha-MSH-mimicking peptide library. The library was inspired by the His-Phe-Arg-Trp active sequence present in alpha-MSH. The novel pentapeptide sequence features the core sequence Bz-Gly-His-D-Phe-AA B-AA A-NR 1 R 2, where AA B is a basic amino acid; AA A is an aromatic amino acid and either amide or alkyl-amide C-terminus (Scheme 1).
Scheme 1. Core peptide sequence.
Biological testing of this library led to the identification of a novel highly potent and selective MC1R-agonist peptide with alpha-MSH activity making it a promising pro-pigmenting and anti-aging molecule.
A small library of eight peptides was synthesized and evaluated for their ability to activate the MC1R receptors and induce cAMP production in the recombinant CHO-MC1R cell line using NDP-MSH as control (EC 50: 0.071 nM) (Table 1). EC 50 response varied from 0.041 to 7.51 nM.
Table 1. MC1R activation assay results.
In terms of the structure–activity relationship (SAR), the most active sequence, 1, was as active as NDP-MSH. Exchange of the L-Trp in 1 for a Naphthyl group (2 and 3) also resulted in sub-nanomolar activity, while the incorporation of a D-Phe (6) somewhat reduced activity compared to the most active sequence. Exchange of the basic arginine residue for the shorter basic diamino butyric acid, Dab, also decreased the activity. Peptide 7, which features a novel amino acid derivative that mimics Trp, by incorporating the CH 2-3-indolyl sidechain into an N-substituted glycine residue, was also shown to be active, although with a reduced efficacy compared to 1.
To confirm our binding and activation results on the MC1-receptor in melanocytes, we subjected alpha-MSH-responsive mouse melanoma cells for 72 h to various concentrations of MC1R-agonist peptides (Figure 1). Peptides 1, 2, 3, and 4 reached maximal activity comparable to 100 nM NDP-alpha-MSH at around 12.3 nM. Peptides 6, 8, and 7 showed significantly less activity and reached maximal activity comparable to NDP-alpha-MSH only beyond 111 nM.
Figure 1. Total melanin synthesis in mouse melanoma cells stimulated with MC1R-agonist peptides. MC1R-agonist peptides were incubated at various concentrations together with the near confluent mouse melanoma cell line B16F10. NDP-alpha-MSH at 100 nM was used as the reference and set to 100% melanin (red line). Error bars represent standard error of the mean of three experiments.
To investigate the melanin synthesis activity of the peptides in a more complex system, we incubated peptides 1 and 4 topically on human abdominal skin ex vivo. Peptides 1 and 4 significantly stimulated pigmentation up to 31% (p< 0.05 vs vehicle) compared to vehicle-treated control tissue at 30 µM peptide (Figure 2). Higher concentrations showed a similar activity of up to 21% more pigmentation at 200 µM peptide.
Figure 2. Stimulation of pigmentation by MC1R-agonist peptides ex vivo. Melanin was quantified on skin sections using ImageJ software (NIH, USA). Human abdominal skin was topically treated with peptides at various concentrations for six days. * p< 0.05 vs vehicle by unpaired Student’s t-test. Error bars represent standard error of the mean of twelve samples. Red line indicates baseline pigmentation score (vehicle only).
We were intrigued by the somewhat reverse dose-dependent activity of peptide 4 and tested it at lower concentrations. This revealed an optimal activity for pigmentation enhancement at 30 µM (Figure 3a). The increase in pigmentation was confirmed using Fontana-Masson staining of human abdominal skin sections treated with peptide 4, where a strong black signal indicative of melanocytes making increased amounts of melanin could be seen at the basal membrane (Figure 3b).
Figure 3. (a) The most active peptide ex vivo, 4, had an optimal activity at 30 µM. * p< 0.05 by unpaired Student’s t-test. Error bars represent standard error of the mean of twelve samples. (b) Representative sections of human abdominal skin. Fontana-Masson staining shows black melanin in melanocytes at the basal membrane. Red line indicates baseline pigmentation score (vehicle only).
As peptide entry 4 displayed the highest activity ex vivo, we were interested if its activation of MC1R was specific. To this end, we tested activation of various GPCRs. We selected these GPCRs based on their cellular functions related to MC1R (see Discussion for details). The results of this investigation are shown in Table 2 and they revealed a specific activity of pentapeptide 4 on MC1R with respect to the tested receptors.
Table 2. EC 50 values of peptide 4 on all GPCRs tested. EC 50 values were determined using eight concentrations in a range from 0.01 to 100,000 nM. EC 50 on MC1R is from Table 1 and shown for comparison. For abbreviations and explanations on the various receptors see Discussion.
As peptide 4 was able to induce pigmentation in human skin ex vivo, we were interested if it was able to induce the expression
