Synergistic Potential of α-Melanocyte Stimulating Hormone Based Analogues with Conventional Antibiotic against Planktonic, Biofilm-Embedded, and Systemic Infection Model of MRSA.

The repotentiation of the existing antibiotics by exploiting the combinatorial potential of antimicrobial peptides (AMPs) with them is a promising approach to address the challenges of slow antibiotic development and rising antimicrobial resistance. In the current study, we explored the ability of lead second generation Ana-peptides viz. Ana-9 and Ana-10, derived from Alpha-Melanocyte Stimulating Hormone (α-MSH), to act synergistically with different classes of conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The peptides exhibited prominent synergy with ß-lactam antibiotics, namely, oxacillin, ampicillin, and cephalothin, against planktonic MRSA. Furthermore, the lead combination of Ana-9/Ana-10 with oxacillin provided synergistic activity against clinical MRSA isolates. Though the treatment of MRSA is complicated by biofilms, the lead combinations successfully inhibited biofilm formation and also demonstrated biofilm disruption potential. Encouragingly, the peptides alone and in combination were able to elicit in vivo anti-MRSA activity and reduce the bacterial load in the liver and kidney of immune-compromised mice. Importantly, the presence of Ana-peptides at sub-MIC doses slowed the resistance development against oxacillin in MRSA cells. Thus, this study highlights the synergistic activity of Ana-peptides with oxacillin advocating for the potential of Ana-peptides as an alternative therapeutic and could pave the way for the reintroduction of less potent conventional antibiotics into clinical use against MRSA infections.

Evaluation of the therapeutic efficacy of 213Bi-labelled DOTA-conjugated alpha-melanocyte stimulating hormone peptide analogues in melanocortin-1 receptor positive preclinical melanoma model.

Melanocortin-1 receptor (MC1-R) targeting alpha-melanocyte stimulating hormone-analogue (α-MSH) biomolecules labelled with α-emitting radiometal seem to be valuable in the targeted radionuclide therapy of MC1-R positive melanoma malignum (MM). Herein is reported the anti-tumor in vivo therapeutic evaluation of MC1-R-affine [213Bi]Bi-DOTA-NAPamide and HOLDamide treatment in MC1-R positive B16-F10 melanoma tumor-bearing C57BL/6J mice. On the 6th, 8th and 10th days post tumor cell inoculation; the treated groups of mice were intravenously injected with approximately 5 MBq of both amide derivatives. Beyond body weight and tumor volume assessment, [68Ga]Ga-DOTA-HOLDamide and NAPamide-based PET/MRI scans, and ex vivo biodistribution studies were executed 30,- and 90 min postinjection. In the PET/MRI imaging studies the B16-F10 tumors were clearly visualized with both 68Ga-labelled tracers, however, significantly lower tumor-to-muscle (T/M) ratios were observed by using [68Ga]Ga-DOTA-HOLDamide. After alpha-radiotherapy treatment the tumor size of the control group was larger relative to both treated cohorts, while the smallest tumor volumes were observed in the NAPamide-treated subclass on the 10th day. Relatively higher [213Bi]Bi-DOTA-NAPamide accumulation in the B16-F10 tumors (%ID/g: 2.71 ± 0.15) with discrete background activity led to excellent T/M ratios, particularly 90 min postinjection. Overall, the therapeutic application of receptor selective [213Bi]Bi-DOTA-NAPamide seems to be feasible in MC1-R positive MM management.

In Vivo evaluation of newly synthesized 213Bi-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptide analogues in melanocortin-1 receptor (MC1-R) positive experimental melanoma model.

Given the rising pervasiveness of melanocortin-1 receptor (MC1-R) positive melanoma malignum (MM) and pertinent metastases, radiolabelled receptor-affine alpha-melanocyte stimulating hormone-analogue (α-MSH analogue) imaging probes would be of crucial importance in timely tumor diagnostic assessment. Herein we aimed at investigating the biodistribution and the MM targeting potential of newly synthesized 213Bi-conjugated MC1-R specific peptide-based radioligands with the establishment of MC1-R overexpressing MM preclinical model. DOTA-conjugated NAP, -HOLD, -FOLD, -and MARSamide were labelled with 213Bi. Ex vivo biodistribution studies were conducted post-administration of 3.81 ± 0.32 MBq [213Bi]Bi-DOTA conjugated deriva-tives into twenty B16-F10 tumor-bearing C57BL/6 J and healthy mice. Organ Level Internal Dose Assessment (OLINDA) and IDAC-Dose were used to calculate translational data-based absorbed radiation dose in human organs. Moderate or low %ID/g uptake of [213Bi]Bi-DOTA conjugated NAP, -HOLD, -and MARSamide and significantly increased [213Bi]Bi-DOTA-FOLDamide accumulation was observed in the thoracic and abdominal organs (p ≤ 0.01). High [213Bi]Bi-DOTA-NAP (%ID/g:3.76 ± 0.96), -and FOLDamide (%ID/g:3.28 ± 0.95) tumor tracer activity confirmed their MC1-R-affinity. The bladder wall received the highest radiation absorbed dose followed by the kidneys (bladder wall: 1.95·10-2 and 8.97·10-2 mSv/MBq; kidneys: 7.47·10-3 vs. 5.88·10-2 mSv/MBq measured by IDAC and OLINDA; respectively) indicating the suitability of the NAPamide derivative for clinical use. These novel [213Bi]Bi-DOTA-linked peptide probes displaying meaningful MC1-R affinity could be promising molecular probes in MM imaging.

Short Communication: Identification of equine corticotropin-like intermediate lobe peptide (CLIP) binding to an adrenocortipcotrophic hormone (ACTH) assay capture antibody.

A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH, α-melanocyte stimulating hormone (α-MSH, ACTH 1-17) and CLIP. Synthetic ACTH and CLIP bound to the capture antibody of the chemiluminescent ACTH assay, but α-MSH did not. Equine endogenous CLIP was detected by LCMS in pony plasma taken in the autumn and could be eluted from the capture antibody of the ACTH chemiluminescent immunoassay. Further research is required to enable quantification of CLIP. Equine CLIP may alter measured ACTH concentrations in vivo.

Ligands for Melanocortin Receptors: Beyond Melanocyte-Stimulating Hormones and Adrenocorticotropin.

The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands (lipocalin 2, ß-defensin, small molecules, and pharmacoperones), and clinically approved ligands (ACTH, setmelanotide, bremelanotide, and several repurposed drugs).

A basal actinopterygian melanocortin receptor: Molecular and functional characterization of an Mc2r ortholog from the Senegal bichir (Polypterus senegalus).

In bony vertebrates, melanocortin 2 receptor (Mc2r) specifically binds adrenocorticotropic hormone (ACTH) and is responsible for mediating anterior pituitary signaling that stimulates corticosteroid production in the adrenal gland/interrenal cells. In bony fishes Mc2r requires the chaperoning of an accessory protein (Mrap1) to traffic to the membrane surface and bind ACTH. Here, we evaluated the structure and pharmacological properties of Mc2r from the Senegal bichir (Polypterus senegalus), which represents the most basal bony fish from which an Mc2r has been pharmacologically studied to date. In our experiments, cDNA constructs of the Mc2r from the Senegal bichir (sbMc2r) and various vertebrate Mrap1s were heterologously co-expressed in Chinese hamster ovary (CHO) cells, stimulated by ACTH or melanocyte-stimulating hormone (α-MSH) ligands, and assessed using a luciferase reporter gene assay. When expressed without an Mrap1, sbMc2r was not activated by ACTH. When co-expressed with Mrap1 from either chicken (Gallus gallus) or bowfin (Amia calva), sbMc2r could be activated in a dose-dependent manner by ACTH, but not α-MSH. Co-expression of sbMrap2 with sbMc2r resulted in no detectable activation of the receptor. Collectively, these results demonstrate that sbMc2r has pharmacological properties similar to those of Mc2rs of later-evolved bony fishes, such as Mrap1 dependence and ACTH selectivity, indicating that these qualities of Mc2r function are ancestral to all bony fish Mc2rs.

α-Melanocyte-Stimulating Hormone Triggers Melanogenesis Via Activation of the Aryl Hydrocarbon Receptor Pathway in B16F10 Mouse Melanoma Cells.

Melanin is a group of natural pigments that determines the human skin color and provides fundamental protection against the harmful impacts of physical and chemical stimuli. The aim of this study was to establish the regulatory role of aryl hydrocarbon receptor (AhR) in α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis. In the present study, following knockdown of AhR, murine B16F10 cells were treated with α-MSH (200 nM) and tyrosinase activities, cellular melanin content, mRNA levels of several important genes involved in melanogenesis including AhR, CTNNB1, TYR2, and microphthalmia-associated transcription factor (MITF) were measured as endpoints. Exposure to α-MSH led to elevated expression of AhR, CTNNB1, MITF, and TYR in accordance with increased tyrosinase enzyme activity as well as a significant rise in the total melanin content. Our results suggest that AhR plays a regulatory role in α-MSH-stimulated melanogenesis.

Pharmacological characterization of three chicken melanocortin-3 receptor mutants.

The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor and potentially important in production traits. Three naturally occurring mutations (M54L, G104S, and L151R) in chicken MC3R (cMC3R) were reported previously to be associated with production traits. Here, we inserted the full-length cMC3R coding sequence into pcDNA3.1(+) and generated the 3 mutations by site-directed mutagenesis. The total and cell surface expression of the receptors was measured by flow cytometry. We analyzed the pharmacological characteristics, including binding and cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase (MAPK) signaling, using 6 ligands ([Nle4, D-Phe7]-α-melanocyte stimulating hormone (MSH), α-, ß-, γ-, and D-Trp8-γ-MSHs, and agouti-related peptide). All mutants had similar total and cell surface expression as the wild-type (WT) cMC3R. M54L had similar pharmacological properties as the WT cMC3R. G104S did not exhibit any specific binding but had minimal response to α-, ß-, γ-, and D-Trp8-γ-MSH, although it generated 24% WT response when stimulated by NDP-MSH. Although L151R had normal binding, the responses to agonists were reduced to approximately 25% of that of the WT. In MAPK signaling, all 3 mutants showed significantly increased agonist-stimulated phosphorylation of extracellular signal-regulated protein kinases 1/2, indicating the existence of biased signaling at G104S and L151R. In summary, our studies demonstrated that although all 3 mutations are significantly associated with production traits, only G104S and L151R had severe defects in receptor pharmacology. How M54L might cause production trait differences remains to be investigated.

Activation of Melanocortin Receptors as a Potential Strategy to Reduce Local and Systemic Reactions Induced by Respiratory Viruses.

The clinical hallmarks of infections caused by critical respiratory viruses consist of pneumonia, which can progress to acute lung injury (ALI), and systemic manifestations including hypercoagulopathy, vascular dysfunction, and endotheliitis. The disease outcome largely depends on the immune response produced by the host. The bio-molecular mechanisms underlying certain dire consequences of the infection partly arise from an aberrant production of inflammatory molecules, an event denoted as "cytokine storm". Therefore, in addition to antiviral therapies, molecules able to prevent the injury caused by cytokine excess are under investigation. In this perspective, taking advantage of melanocortin peptides and their receptors, components of an endogenous modulatory system that exerts marked anti-inflammatory and immunomodulatory influences, could be an effective therapeutic strategy to control disease evolution. Exploiting the melanocortin system using natural or synthetic ligands can form a realistic basis to counteract certain deleterious effects of respiratory virus infections. The central and peripheral protective actions exerted following melanocortin receptor activation could allow dampening the harmful events that trigger the cytokine storm and endothelial dysfunction while sustaining the beneficial signals required to elicit repair mechanisms. The long standing evidence for melanocortin safety encourages this approach.

Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers.

Activation of the ARCPOMC→MeA Projection Reduces Food Intake.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) plays an essential role in the control of food intake and energy expenditure. Melanocortin-4 receptors (MC4Rs) are expressed in key areas that are implicated in regulating energy homeostasis. Although the importance of MC4Rs in the paraventricular hypothalamus (PVH) has been well documented, the role of MC4Rs in the medial amygdala (MeA) on feeding remains controversial. In this study, we specifically examine the role of a novel ARCPOMC→MeA neural circuit in the regulation of short-term food intake. To map a local melanocortinergic neural circuit, we use monosynaptic anterograde as well as retrograde viral tracers and perform double immunohistochemistry to determine the identity of the neurons receiving synaptic input from POMC neurons in the ARC. To investigate the role of the ARCPOMC→MeA projection on feeding, we optogenetically stimulate channelrhodopsin-2 (ChR2)-expressing POMC fibers in the MeA. Anterograde viral tracing studies reveal that ARC POMC neurons send axonal projections to estrogen receptor-α (ER-α)- and MC4R-expressing neurons in the MeA. Retrograde viral tracing experiments show that the neurons projecting to the MeA is located mainly in the lateral part of the ARC. Optogenetic stimulation of the ARCPOMC→MeA pathway reduces short-term food intake. This anorectic effect is blocked by treatment with the MC4R antagonist SHU9119. In addition to the melanocortinergic local circuits within the hypothalamus, this extrahypothalamic ARCPOMC→MeA neural circuit would play a role in regulating short-term food intake.

A proopiomelanocortin-derived peptide sequence enhances plasma stability of peptide drugs.

Bioactive peptide drugs hold promise for therapeutic application due to their high potency and selectivity but display short plasma half-life. Examination of selected naturally occurring peptide hormones derived from proteolytic cleavage of the proopiomelanocortin (POMC) precursor lead to the identification of significant plasma-stabilizing properties of a 12-amino acid serine-rich orphan sequence NSSSSGSSGAGQ in human γ3-melanocyte-stimulating hormone (MSH) that is homologous to previously discovered NSn GGH (n = 4-24) sequences in owls. Notably, transfer of this sequence to des-acetyl-α-MSH and the therapeutically relevant peptide hormones neurotensin and glucagon-like peptide-1 likewise enhance their plasma stability without affecting receptor signaling. The stabilizing effect of the sequence module is independent of plasma components, suggesting a direct effect in cis. This natural sequence module may provide a possible strategy to enhance plasma stability, complementing existing methods of chemical modification.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

The Influence of Different Spacers on Biological Profile of Peptide Radiopharmaceuticals for Diagnosis and Therapy of Human Cancers.

BACKGROUND: Cancer is the leading cause of death worldwide. Early detection can reduce the disadvantageous effects of diseases and the mortality in cancer. Nuclear medicine is a powerful tool that has the ability to diagnose malignancy without harming normal tissues. In recent years, radiolabeled peptides have been investigated as potent agents for cancer detection. Therefore, it is necessary to modify radiopeptides in order to achieve more effective agents. OBJECTIVE: This review describes modifications in the structure of radioconjugates with spacers who have improved the specificity and sensitivity of the peptides that are used in oncologic diagnosis and therapy. METHODS: To improve the biological activity, researchers have conjugated these peptide analogs to different spacers and bifunctional chelators. Many spacers of different kinds, such as hydrocarbon chain, amino acid sequence, and poly (ethyleneglycol) were introduced in order to modify the pharmacokinetic properties of these biomolecules. RESULTS: Different spacers have been applied to develop radiolabeled peptides as potential tracers in nuclear medicine. Spacers with different charge and hydrophilicity affect the characteristics of peptide conjugate. For example, the complex with uncharged and hydrophobic spacers leads to increased liver uptake, while the composition with positively charged spacers results in high kidney retention. Therefore, the pharmacokinetics of radio complexes correlates to the structure and total charge of the conjugates. CONCLUSION: Radio imaging technology has been successfully applied to detect a tumor in the earliest stage. For this purpose, the assessment of useful agents to diagnose the lesion is necessary. Developing peptide radiopharmaceuticals using spacers can improve in vitro and in vivo behavior of radiotracers leading to better noninvasive detection and monitoring of tumor growth.

AQB-565 shows promise in preclinical testing in the model of epileptic spasms during infancy: Head-to-head comparison with ACTH.

Epileptic spasms during infancy (infantile spasms) represent a serious treatment and social problem despite their rare occurrence. Current treatments include hormonal therapy (adrenocorticotropin-ACTH or corticosteroids) or vigabatrin (per se or in the combination). These treatments are partially effective and with potentially significant adverse effects. Thus, the search for new effective drugs is warranted. We tested efficacy of a novel fusion peptide AQB-565 developed by Aequus Biopharma in a model of infantile spasms consisting of prenatal exposure to betamethasone and repeated postnatal trigger of spasms with N-methyl-d-aspartic acid (NMDA). AQB-565 molecule includes the first 24 amino acids of ACTH, a ten amino acid linker and a modified melanocyte-stimulating hormone molecule. In contrast to ACTH with almost uniform activity over all peripheral and central melanocortin receptor isoforms, AQB is preferentially active on central melanocortin receptors MC3 and MC4. Here, we used equivalent doses of rat ACTH (full molecule) and AQB-565 and compared their efficacy in a prospective randomized test against of repeated bouts of spasms on postnatal days (P)12, P13 and P15 in the rat model. All doses of ACTH (range 0.02-1.0 mg/kg s.c.) and all doses but one of AQB-565 in the same range suppressed spasms in P15 rats (treatment stopped on P14). There was no dose-dependent effect and both compounds had all-or-none effect that is similar to clinical outcome of hormonal treatment of infantile spasms in children. Thus, AQB-565 may represent a novel treatment of infantile spasms similarly effective as ACTH but with potentially limited side effects.

Immunoregulatory Effects of Neuropeptides on Endothelial Cells: Relevance to Dermatological Disorders.

Many skin diseases, including psoriasis and atopic dermatitis, have a neurogenic component. In this regard, bidirectional interactions between components of the nervous system and multiple target cells in the skin and elsewhere have been receiving increasing attention. Neuropeptides released by sensory nerves that innervate the skin can directly modulate functions of keratinocytes, Langerhans cells, dermal dendritic cells, mast cells, dermal microvascular endothelial cells and infiltrating immune cells. As a result, neuropeptides and neuropeptide receptors participate in a complex, interdependent network of mediators that modulate the skin immune system, skin inflammation, and wound healing. In this review, we will focus on recent studies demonstrating the roles of α-melanocyte-stimulating hormone, calcitonin gene-related peptide, substance P, somatostatin, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and nerve growth factor in modulating inflammation and immunity in the skin through their effects on dermal microvascular endothelial cells.

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.

Novel (1E,3E,5E)-1,6-bis(Substituted phenyl)hexa-1,3,5-triene Analogs Inhibit Melanogenesis in B16F10 Cells and Zebrafish.

The present study aimed to evaluate the anti-melanogenic activity of 1,6-diphenyl-1,3,5-hexatriene and its derivatives in B16F10 murine melanoma cells and zebrafish embryos. Twenty five (1E,3E,5E)-1,6-bis(substituted phenyl)hexa-1,3,5-triene analogs were synthesized and their non-cytotoxic effects were predictively analyzed using three-dimensional quantitative structure-activity relationship approach. Inhibitory activities of these synthetic compounds against melanin synthesis were determined by evaluating melanin content and melanogenic regulatory enzyme expression in B16F10 cells. The anti-melanogenic activity was verified by observing body pigmentation in zebrafishes treated with these compounds. Compound #2, #4, and #6 effectively decreased melanogenesis induced by α-melanocyte-stimulating hormone. In particular, compound #2 remarkably lowered the mRNA and protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and TYRP2 in B16F10 cells and substantially reduced skin pigmentation in the developed larvae of zebrafish. These findings suggest that compound #2 may be used as an anti-melanogenic agent for cosmetic purpose.

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.

The thermogenic effect of nesfatin-1 requires recruitment of the melanocortin system.

Nesfatin-1 is a bioactive polypeptide expressed both in the brain and peripheral tissues and involved in the control of energy balance by reducing food intake. Central administration of nesfatin-1 significantly increases energy expenditure, as demonstrated by a higher dry heat loss; yet, the mechanisms underlying the thermogenic effect of central nesfatin-1 remain unknown. Therefore, in this study, we sought to investigate whether the increase in energy expenditure induced by nesfatin-1 is mediated by the central melanocortin pathway, which was previously reported to mediate central nesfatin-1´s effects on feeding and numerous other physiological functions. With the application of direct calorimetry, we found that intracerebroventricular nesfatin-1 (25 pmol) treatment increased dry heat loss and that this effect was fully blocked by simultaneous administration of an equimolar dose of the melanocortin 3/4 receptor antagonist, SHU9119. Interestingly, the nesfatin-1-induced increase in dry heat loss was positively correlated with body weight loss. In addition, as assessed with thermal imaging, intracerebroventricular nesfatin-1 (100 pmol) increased interscapular brown adipose tissue (iBAT) as well as tail temperature, suggesting increased heat production in the iBAT and heat dissipation over the tail surface. Finally, nesfatin-1 upregulated pro-opiomelanocortin and melanocortin 3 receptor mRNA expression in the hypothalamus, accompanied by a significant increase in iodothyronine deiodinase 2 and by a nonsignificant increase in uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha mRNA in the iBAT. Overall, we clearly demonstrate that nesfatin-1 requires the activation of the central melanocortin system to increase iBAT thermogenesis and, in turn, overall energy expenditure.