α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor.

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.

Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver.

Melanocortin 1 receptor (MC1-R) is widely expressed in melanocytes and leukocytes and is thus strongly implicated in the regulation of skin pigmentation and inflammation. MC1-R has also been found in the rat and human liver, but its functional role has remained elusive. We hypothesized that MC1-R is functionally active in the liver and involved in the regulation of cholesterol and bile acid metabolism. We generated hepatocyte-specific MC1-R knock-out (Mc1r LKO) mice and phenotyped the mouse model for lipid profiles, liver histology, and bile acid levels. Mc1r LKO mice had significantly increased liver weight, which was accompanied by elevated levels of total cholesterol and triglycerides in the liver as well as in the plasma. These mice demonstrated also enhanced liver fibrosis and a disturbance in bile acid metabolism as evidenced by markedly reduced bile acid levels in the plasma and feces. Mechanistically, using HepG2 cells as an in vitro model, we found that selective activation of MC1-R in HepG2 cells reduced cellular cholesterol content and enhanced uptake of low- and high-density lipoprotein particles via a cAMP-independent mechanism. In conclusion, the present results demonstrate that MC1-R signaling in hepatocytes regulates cholesterol and bile acid metabolism and its deficiency leads to hypercholesterolemia and enhanced lipid accumulation and fibrosis in the liver.

Ψ and χ Angle Constrains at the C-Terminus Trp Position of the Melanotropin Tetrapeptide Ac-His-d-Phe-Arg-Trp-NH2 Lead to Potent and Selective Agonists at hMC1R and hMC4R.

Melanocortin receptors (MCRs) are a family of G protein-coupled receptors that regulate important physiological functions. Yet, drug development targeting MCRs is hindered by potential side effects due to a lack of receptor subtype-selective ligands with bioavailability. Here, we report novel synthetic pathways to introduce Ψ and χ angle constraints at the C-terminus Trp position of the nonselective prototype tetrapeptide agonist Ac-His-d-Phe-Arg-Trp-NH2. With these conformational constraints, peptide 1 (Ac-His-d-Phe-Arg-Aia) shows improved selectivity at hMC1R, with an EC50 of 11.2 nM for hMC1R and at least 15-fold selectivity compared to other MCR subtypes. Peptide 3 (Ac-His-pCF3-d-Phe-Arg-Aia) is a potent and selective hMC4R agonist with an EC50 of 4.1 nM at hMC4R and at least ninefold selectivity. Molecular docking studies reveal that the Ψ and χ angle constraints force the C-terminal Aia residue to flip and interact with TM6 and TM7, a feature that we hypothesize leads to the receptor subtype selectivity.

MC1R Peptide Agonist Self-Assembles into a Hydrogel That Promotes Skin Pigmentation for Treating Vitiligo.

Vitiligo, a common skin disease that seriously affects 0.5-2.0% of the worldwide population, lacks approved therapeutics due to a wide range of adverse side effects. As a key regulator of skin pigmentation, MC1R may be an effective therapeutic target for vitiligo. Herein, we report an MC1R peptide agonist that directly self-assembles into nanofibrils that form a hydrogel matrix under normal physiological conditions. This hydrogel exhibits higher stability than free peptides, sustained release, rapid recovery from shear-thinning, and resistance to enzymatic proteolysis. Furthermore, this peptidal MC1R agonist upregulates tyrosinase, tyrosinase-related protein-1 (TYRP-1), and tyrosinase-related protein-2 (TYRP-2) to stimulate melanin synthesis. More importantly, MC1R agonist hydrogel promotes skin pigmentation in mice more potently than free MC1R agonist. This study supports the development of this MC1R agonist hydrogel as a promising pharmacological intervention for vitiligo.

CLIPSing Melanotan-II to Discover Multiple Functionally Selective hMCR Agonists.

The pleiotropic role played by melanocortin receptors (MCRs) in both physiological and pathological processes has stimulated medicinal chemists to develop synthetic agonists/antagonists with improved potency and selectivity. Here, by deploying the Chemical Linkage of Peptide onto Scaffolds strategy, we replaced the lactam cyclization of melanotan II (MT-II), a potent and unselective agonist of human MCRs (hMCRs), with different xylene-derived thioethers. The newly designed peptides displayed binding affinities toward MCRs ranging from the low nanomolar to the sub-micromolar range, highlighting a correlation between the explored linkers and the affinity toward hMCRs. In contrast to the parent peptide (MT-II), compound 5 displayed a remarkable functional selectivity toward the hMC1R. Enhanced sampling molecular dynamics simulations were found to be instrumental in outlining how the employed cyclization strategy affects the peptides' conformational behavior and, as a consequence, the detected hMC1R affinity. Additionally, a model of the peptide 5/hMC1R complex employing the very recently reported cryogenic electron microscopy receptor structure was provided.

Aged Brains Express Less Melanocortin Receptors, Which Correlates with Age-Related Decline of Cognitive Functions.

Brain G-protein coupled receptors have been hypothesized to be potential targets for maintaining or restoring cognitive function in normal aged individuals or in patients with neurodegenerative disease. A number of recent reports suggest that activation of melanocortin receptors (MCRs) in the brain can significantly improve cognitive functions of normal rodents and of different rodent models of the Alzheimer's disease. However, the potential impact of normative aging on the expression of MCRs and their potential roles for modulating cognitive function remains to be elucidated. In the present study, we first investigated the expression of these receptors in six different brain regions of young (6 months) and aged (23 months) rats following assessment of their cognitive status. Correlation analysis was further performed to reveal potential contributions of MCR subtypes to spatial learning and memory. Our results revealed statistically significant correlations between the expression of several MCR subtypes in the frontal cortex/hypothalamus and the hippocampus regions and the rats' performance in spatial learning and memory only in the aged rats. These findings support the hypothesis that aging has a direct impact on the expression and function of MCRs, establishing MCRs as potential drug targets to alleviate aging-induced decline of cognitive function.

Multiple Applications of a Novel Biarsenical Imaging Probe in Fluorescence and PET Imaging of Melanoma.

A new fluorescent biarsenical peptide labeling probe was synthesized and labeled with the radioactive isotopes 11C and 18F. The utility of this probe was demonstrated by installing each of these isotopes into a melanocortin 1 receptor (MC1R) binding peptide, which targets melanoma tumors. Its applicability was further showcased by subsequent in vitro imaging in cells as well as in vivo imaging in melanoma xenograft mice by fluorescence and positron emission tomography.

Development of Ligand-Drug Conjugates Targeting Melanoma through the Overexpressed Melanocortin 1 Receptor.

Melanoma is a lethal form of skin cancer. Despite recent breakthroughs of BRAF-V600E and PD-1 inhibitors showing remarkable clinical responses, melanoma can eventually survive these targeted therapies and become resistant. To solve the drug resistance issue, we designed and synthesized ligand-drug conjugates that couple cytotoxic drugs, which have a low cancer resistance issue, with the melanocortin 1 receptor (MC1R) agonist melanotan-II (MT-II), which provides specificity to MC1R-overexpressing melanoma. The drug-MT-II conjugates maintain strong binding interactions to MC1R and induce selective drug delivery to A375 melanoma cells through its MT-II moiety in vitro. Furthermore, using camptothecin as the cytotoxic drug, camptothecin-MT-II (compound 1) can effectively inhibit A375 melanoma cell growth with an IC50 of 16 nM. By providing selectivity to melanoma cells through its MT-II moiety, this approach of drug-MT-II conjugates enables us to have many more options for cytotoxic drug selection, which can be the key to solving the cancer resistant problem for melanoma.

Melanocortin 3 receptor activation with [D-Trp8]-γ-MSH suppresses inflammation in apolipoprotein E deficient mice.

The melanocortin MC1 and MC3 receptors elicit anti-inflammatory actions in leukocytes and activation of these receptors has been shown to alleviate arterial inflammation in experimental atherosclerosis. Thus, we aimed to investigate whether selective targeting of melanocortin MC3 receptor protects against atherosclerosis. Apolipoprotein E deficient (ApoE-/-) mice were fed high-fat diet for 12 weeks and randomly assigned to receive either vehicle (n = 11) or the selective melanocortin MC3 receptor agonist [D-Trp(8)]-gamma-melanocyte-stimulating hormone ([D-Trp8]-γ-MSH; 15 µg/day, n = 10) for the last 4 weeks. Lesion size as well as macrophage and collagen content in the aortic root plaques were determined. Furthermore, leukocyte counts in the blood and aorta and cytokine mRNA expression levels in the spleen, liver and aorta were quantified. No effect was observed in the body weight development or plasma cholesterol level between the two treatment groups. However, [D-Trp8]-γ-MSH treatment significantly reduced plasma levels of chemokine (C-C motif) ligands 2, 4 and 5. Likewise, cytokine and adhesion molecule expression levels were reduced in the spleen and liver of γ-MSH-treated mice, but not substantially in the aorta. In line with these findings, [D-Trp8]-γ-MSH treatment reduced leukocyte counts in the blood and aorta. Despite reduced inflammation, [D-Trp8]-γ-MSH did not change lesion size, macrophage content or collagen deposition of aortic root plaques. In conclusion, the findings indicate that selective activation of melanocortin MC3 receptor by [D-Trp8]-γ-MSH suppresses systemic and local inflammation and thereby also limits leukocyte accumulation in the aorta. However, the treatment was ineffective in reducing atherosclerotic plaque size.

Development of N-Acetylated Dipalmitoyl-S-Glyceryl Cysteine Analogs as Efficient TLR2/TLR6 Agonists.

Cancer vaccine is a promising immunotherapeutic approach to train the immune system with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in cancer vaccines to mimic an infection process and amplify immune responses. The Toll-like receptor 2 and 6 (TLR2/TLR6) agonist dipalmitoyl-S-glyceryl cysteine (Pam2Cys) was demonstrated as an ideal candidate for synthetic vaccine adjuvants. However, the synthesis of Pam2Cys requires expensive N-protected cysteine as a key reactant, which greatly limits its application as a synthetic vaccine adjuvant in large-scaled studies. Here, we report the development of N-acetylated Pam2Cys analogs as TLR2/TLR6 agonists. Instead of N-protected cysteine, the synthesis utilizes N-acetylcysteine to bring down the synthetic costs. The N-acetylated Pam2Cys analogs were demonstrated to activate TLR2/TLR6 in vitro. Moreover, molecular docking studies were performed to provide insights into the molecular mechanism of how N-acetylated Pam2Cys analogs bind to TLR2/TLR6. Together, these results suggest N-acetylated Pam2Cys analogs as inexpensive and promising synthetic vaccine adjuvants to accelerate the development of cancer vaccines in the future.

Replacement of Arg with Nle and modified D-Phe in the core sequence of MSHs, Ac-His-D-Phe-Arg-Trp-NH2, leads to hMC1R selectivity and pigmentation.

Melanoma skin cancer is the fastest growing cancer in the US [1]. A great need exists for improved formulations and mechanisms to prevent and protect human skin from cancers and other skin damage caused by sunlight exposure. Current efforts to prevent UV damage to human skin, which in many cases leads to melanoma and other skin cancers. The primordial melanocortin-1 receptor (MC1R) is involved in regulating skin pigmentation and hair color, which is a natural prevention from UV damage. The endogenous melanocortin agonists induce pigmentation and share a core pharmacophore sequence "His-Phe-Arg-Trp", and it was found that substitution of the Phe by D-Phe results in increasing melanocortin receptor potency. To improve the melanocortin 1 receptor (MC1R) selectivity a series of tetra-peptides with the moiety of Ac-Xaa-Yaa-Nle-Trp-NH2, and structural modifications to reduce electrostatic ligand-receptor interactions have been designed and synthesized. It is discovered that the tetrapeptide Ac-His-D-Phe(4-CF3)-Nle-Trp-NH2 resulted in a potent and selective hMC1R agonist at the hMC1R (EC50: 10 nM). Lizard anolis carolinensis pigmentation study shows very high potency in vivo. NMR studies revealed a reversed ß turn structure which led to the potency and selectivity towards the hMC1R.

Development of Macrocyclic Peptidomimetics Containing Constrained α,α-Dialkylated Amino Acids with Potent and Selective Activity at Human Melanocortin Receptors.

We report the development of macrocyclic melanocortin derivatives of MT-II and SHU-9119, achieved by modifying the cycle dimension and incorporating constrained amino acids in ring-closing. This study culminated in the discovery of novel agonists/antagonists with an unprecedented activity profile by adding pieces to the puzzle of the melanocortin receptor selectivity. Finally, the resulting 19- and 20-membered rings represent a suitable frame for the design of further therapeutic ligands as selective modulators of the melanocortin system.

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1.

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p Ki of 8.73 ± 0.08 ( Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation.

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

Novel approaches to the design of bioavailable melanotropins.

INTRODUCTION: The melanocortin system is a primordial and critical system for survival, involved in a wide variety of physiological functions. It includes melanocortin receptors (MCRs) and melanotropin ligands (MCLs). MCRs are important drug targets that can regulate several key physiological processes. Extensive efforts have been made to develop peptide and peptidomimetics targeting melanocortin receptors including MC1R, MC3R, MC4R and MC5R. Most research is focused on developing potent and selective melanotropins. However, developing bioavailable melanotropins remains challenging. Areas covered: Herein, the authors summarize promising strategies for developing bioavailable MCLs by using cyclized N-methylated melanotropins, and using cyclotide and tetrapeptide as templates. They discuss their unique advantages in oral availability and targeting MCRs in the central nervous system or in peripheral tissues. Finally, they discuss the observed differences in thepharmacology of MCRs between in vitro and in vivo tests. Expert opinion: N-methylated cyclized melanotropins have great potential to become bio- available drugs targeting MCRs in the brain, while MCR-grafted cyclotides tend to target MCRs in peripheral tissue. A better understanding of the biased signaling process is a new challenge and opportunity for the future discovery of bioavailable MCLs.

Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His6-d-Nal(2')7-NMe-Arg8-Trp9-Lys]-NH2 (15) and Ac-Nle-c[Asp-His6-d-Nal(2')7-NMe-Arg8-NMe-Trp9-NMe-Lys]-NH2 (17). It is known that the pharmacophore (His6-DNal7-Arg8-Trp9) of the SHU-9119 peptides occupies a ß II-turn-like region with the turn centered about DNal7-Arg8. The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg8 and Trp9 side chains are involved in a majority of the interactions with the receptor. While Arg8 forms polar contacts with D154 and D158 of hMC3R, Trp9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp9-hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

BACKGROUND: The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. METHODS: Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. RESULTS: Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. CONCLUSIONS: Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse cholesterol transport.

Design of cyclized selective melanotropins.

This article describes the development of cyclic peptides for G-protein coupled receptors to enable structure-function knowledge and the design of novel therapeutics. One important property of cyclic peptides is that they tend to be resistant to the digestion, enabling them to survive in the human digestive tract. This trait makes them very important as drug leads or as scaffolds which, in theory, can be engineered to incorporate a peptide domain of medicinal value. This is especially important for delivery of peptides that would be destroyed without such implementation. The melanocortin system is the focus of this article, and includes melanotropin ligands and melanocortin receptors. We examine two strategies to constrain the melanotropin peptide backbone. The first is based on global constraint of peptides by cyclization using various kinds of linkers. In the second approach we describe the use of a natural cyclized template, the cyclotide, to graft the melanotropin phamacophore, -His-Phe-Arg-Trp-, to obtain selective drug leads. In these examples the conserved melanocyte stimulating hormone pharmacophore is examined and the modified peptides were synthesized by solid phase methodology. Biological studies confirmed the production of selective, potent and in some cases orally available ligands.

Characterization of TCP-1 probes for molecular imaging of colon cancer.

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.