Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.

Exploring FDA-Approved Frontiers: Insights into Natural and Engineered Peptide Analogues in the GLP-1, GIP, GHRH, CCK, ACTH, and α-MSH Realms.

Peptides continue to gain significance in the pharmaceutical arena. Since the unveiling of insulin in 1921, the Food and Drug Administration (FDA) has authorised around 100 peptides for various applications. Peptides, although initially derived from endogenous sources, have evolved beyond their natural origins, exhibiting favourable therapeutic effectiveness. Medicinal chemistry has played a pivotal role in synthesising valuable natural peptide analogues, providing synthetic alternatives with therapeutic potential. Furthermore, key chemical modifications have enhanced the stability of peptides and strengthened their interactions with therapeutic targets. For instance, selective modifications have extended their half-life and lessened the frequency of their administration while maintaining the desired therapeutic action. In this review, I analyse the FDA approval of natural peptides, as well as engineered peptides for diabetes treatment, growth-hormone-releasing hormone (GHRH), cholecystokinin (CCK), adrenocorticotropic hormone (ACTH), and α-melanocyte stimulating hormone (α-MSH) peptide analogues. Attention will be paid to the structure, mode of action, developmental journey, FDA authorisation, and the adverse effects of these peptides.

Low resting metabolic rate and increased hunger due to ß-MSH and ß-endorphin deletion in a canine model.

Mutations that perturb leptin-melanocortin signaling are known to cause hyperphagia and obesity, but energy expenditure has not been well studied outside rodents. We report on a common canine mutation in pro-opiomelanocortin (POMC), which prevents production of ß-melanocyte-stimulating hormone (ß-MSH) and ß-endorphin but not α-MSH; humans, similar to dogs, produce α-MSH and ß-MSH from the POMC propeptide, but rodents produce only α-MSH. We show that energy expenditure is markedly lower in affected dogs, which also have increased motivational salience in response to a food cue, indicating increased wanting or hunger. There was no difference in satiety at a modified ad libitum meal or in their hedonic response to food, nor disruption of adrenocorticotropic hormone (ACTH) or thyroid axes. In vitro, we show that ß-MSH signals comparably to α-MSH at melanocortin receptors. These data implicate ß-MSH and ß-endorphin as important in determining hunger and moderating energy expenditure and suggest that this role is independent of the presence of α-MSH.

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.

Molecular mechanisms underlying Tao-Hong-Si-Wu decoction treating hyperpigmentation based on network pharmacology, Mendelian randomization analysis, and experimental verification.

CONTEXT: Hyperpigmentation, a common skin condition marked by excessive melanin production, currently has limited effective treatment options. OBJECTIVE: This study explores the effects of Tao-Hong-Si-Wu decoction (THSWD) on hyperpigmentation and to elucidate the underlying mechanisms. MATERIALS AND METHODS: We employed network pharmacology, Mendelian randomization, and molecular docking to identify THSWD's hub targets and mechanisms against hyperpigmentation. The Cell Counting Kit-8 (CCK-8) assay determined suitable THSWD treatment concentrations for PIG1 cells. These cells were exposed to graded concentrations of THSWD-containing serum (2.5%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%) and treated with α-MSH (100 nM) to induce an in vitro hyperpigmentation model. Assessments included melanin content, tyrosinase activity, and Western blotting. RESULTS: ALB, IL6, and MAPK3 emerged as primary targets, while quercetin, apigenin, and luteolin were the core active ingredients. The CCK-8 assay indicated that concentrations between 2.5% and 20% were suitable for PIG1 cells, with a 50% cytotoxicity concentration (CC50) of 32.14%. THSWD treatment significantly reduced melanin content and tyrosinase activity in α-MSH-induced PIG1 cells, along with downregulating MC1R and MITF expression. THSWD increased ALB and p-MAPK3/MAPK3 levels and decreased IL6 expression in the model cells. DISCUSSION AND CONCLUSION: THSWD mitigates hyperpigmentation by targeting ALB, IL6, and MAPK3. This study paves the way for clinical applications of THSWD as a novel treatment for hyperpigmentation and offers new targeted therapeutic strategies.

Rigid linker peptides improve the stability and anti-inflammation effect of human serum albumin and α-melanocyte-stimulating hormone fusion proteins.

The anti-inflammatory effect of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS) has been reported for 40 years. However, the short half-life of α-MSH limits its clinical applications. The previous study has shown that a fusion protein comprising protein transduction domain (PTD), human serum albumin (HSA), and α-MSH extends the half-life of α-MSH, but its anti-inflammatory effect is not satisfactory. In this study, optimization of the structures of fusion proteins was attempted by changing the linker peptide between HSA and α-MSH. The optimization resulted in the improvement of various important characteristics, especially the stability and anti-inflammatory bioactivity, which are important features in protein medicines. Compared to the original linker peptide L0, the 5-amino-acid rigid linker peptide L6 (PAPAP) is the best option for further investigation due to its higher expression (increased by 6.27%), improved purification recovery (increased by 60.8%), excellent thermal stability (Tm = 83.5°C) and better inhibition in NF-κB expression (increased by 81.5%). From this study, the significance of the design of linker peptides in the study of structure-activity relationship of fusion proteins was proved.

Interruption of p38MAPK-MSK1-CREB-MITF-M pathway to prevent hyperpigmentation in the skin.

Background: Melanocortin 1 receptor (MC1R), a receptor of α-melanocyte-stimulating hormone (α-MSH), is exclusively present in melanocytes where α-MSH/MC1R stimulate melanin pigmentation through microphthalmia-associated transcription factor M (MITF-M). Toll-like receptor 4 (TLR4), a receptor of endotoxin lipopolysaccharide (LPS), is distributed in immune and other cell types including melanocytes where LPS/TLR4 activate transcriptional activity of nuclear factor (NF)-κB to express cytokines in innate immunity. LPS/TLR4 also up-regulate MITF-M-target melanogenic genes in melanocytes. Here, we propose a molecular target of antimelanogenic activity through elucidating inhibitory mechanism on α-MSH-induced melanogenic programs by benzimidazole-2-butanol (BI2B), an inhibitor of LPS/TLR4-activated transcriptional activity of NF-κB. Methods: Ultraviolet B (UV-B)-irradiated skins of HRM-2 hairless mice and α-MSH-activated melanocyte cultures were employed to examine melanogenic programs. Results: Topical treatment with BI2B ameliorated UV-B-irradiated skin hyperpigmentation in mice. BI2B suppressed the protein or mRNA levels of melanogenic markers, such as tyrosinase (TYR), MITF-M and proopiomelanocortin (POMC), in UV-B-exposed and pigmented skin tissues. Moreover, BI2B inhibited melanin pigmentation in UV-B-irradiated co-cultures of keratinocyte and melanocyte cells and that in α-MSH-activated melanocyte cultures. Mechanistically, BI2B inhibited the activation of cAMP response element-binding protein (CREB) in α-MSH-induced melanogenic programs and suppressed the expression of MITF-M at the promoter level. As a molecular target, BI2B primarily inhibited mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)-catalyzed kinase activity on p38MAPK. Subsequently, BI2B interrupted downstream pathway of p38MAPK-mitogen and stress-activated protein kinase-1 (MSK1)-CREB-MITF-M, and suppressed MITF-M-target melanogenic genes, encoding enzymes TYR, TYR-related protein-1 (TRP-1) and dopachrome tautomerase (DCT) in melanin biosynthesis, and encoding proteins PMEL17 and Rab27A in the transfer of pigmented melanosomes to the overlaying keratinocytes in the skin. Conclusion: Targeting the MKK3-p38MAPK-MSK1-CREB-MITF-M pathway was suggested as a rationale to inhibit UV-B- or α-MSH-induced facultative melanogenesis and as a strategy to prevent acquired pigmentary disorders in the skin.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

The Melanocortin and Endorphin Neuropeptides in Patients with Restless Legs Syndrome.

OBJECTIVE: Based upon similarities between the urge to move and sensory discomfort of restless legs syndrome (RLS) and properties of melanocortin hormones, including their incitement of movement and hyperalgesia, we assessed plasma and cerebrospinal fluid (CSF) α-melanocyte-stimulating hormone (α-MSH) and ß-endorphin in RLS patients and controls. METHODS: Forty-two untreated moderate-to-severe RLS patients and 44 matched controls underwent venipuncture at 19:00, 20:30, and 22:00; 37 RLS and 36 controls had lumbar puncture at 21:30. CSF and plasma were analyzed for pro-opiomelanocortin (POMC), adrenocorticotropin hormone (ACTH), α-MSH, ß-MSH, and ß-endorphin by immunoassay. RLS severity was assessed by International RLS Study Group Severity Scale. RESULTS: RLS participants were 52.7 ± 12.0 years old, 61.9% were women, 21.4% had painful RLS, and RLS severity was 24.8 ± 9.0. Controls had similar age and sex. Plasma ACTH, α-MSH, and ß-endorphin were similar between groups. Plasma POMC was significantly greater in RLS than controls (17.0 ± 11.5 vs 12.7 ± 6.1fmol/ml, p = 0.048). CSF ACTH was similar between groups. CSF ß-MSH was significantly higher in painful than nonpainful RLS or controls (48.2 ± 24.8 vs 32.1 ± 14.8 vs 32.6 ± 15.2pg/ml, analysis of variance [ANOVA] p = 0.03). CSF α-MSH was higher in RLS than controls (34.2 ± 40.9 vs 20.3 ± 11.0pg/ml, p = 0.062). CSF ß-EDP was lowest in painful RLS, intermediate in nonpainful RLS, and highest in controls (8.0 ± 3.4 vs 10.8 ± 3.1 vs 12.3 ± 5.0pg/ml, ANOVA p = 0.049). The ratio of the sum of CSF α- and ß-MSH to CSF ß-endorphin was highest, intermediate, and lowest in painful RLS, nonpainful RLS, and controls (p = 0.007). INTERPRETATION: CSF ß-MSH is increased and CSF ß-endorphin decreased in RLS patients with painful symptoms. ANN NEUROL 2024;95:688-699.

2,4,6-Triphenyl-1-hexene, an Anti-Melanogenic Compound from Marine-Derived Bacillus sp. APmarine135.

Although melanin protects against ultraviolet radiation, its overproduction causes freckles and senile lentigines. Recently, various biological effects of metabolites derived from marine microorganisms have been highlighted due to their potential for biological and pharmacological applications. In this study, we discovered the anti-melanogenic effect of Bacillus sp. APmarine135 and verified the skin-whitening effect. Fractions of APmarine135 showed the melanin synthesis inhibition effect in B16 melanoma cells, and 2,4,6-triphenyl-1-hexene was identified as an active compound. The melanogenic capacity of 2,4,6-triphenyl-1-hexene (1) was investigated by assessing the intracellular melanin content in B16 cells. Treatment with 5 ppm of 2,4,6-triphenyl-1-hexene (1) for 72 h suppressed the α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin increase to the same level as in the untreated control group. Additionally, 2,4,6-triphenyl-1-hexene (1) treatment suppressed the activity of tyrosinase, the rate-limiting enzyme for melanogenesis. Moreover, 2,4,6-triphenyl-1-hexene (1) treatment downregulated tyrosinase, Tyrp-1, and Tyrp-2 expression by inhibiting the microphthalmia-associated transcription factor (MITF). Furthermore, 2,4,6-triphenyl-1-hexene (1) treatment decreased the melanin content in the three-dimensional (3D) human-pigmented epidermis model MelanoDerm and exerted skin-whitening effects. Mechanistically, 2,4,6-triphenyl-1-hexene (1) exerted anti-melanogenic effects by suppressing tyrosinase, Tyrp-1, and Tyrp-2 expression and activities via inhibition of the MITF. Collectively, these findings suggest that 2,4,6-triphenyl-1-hexene (1) is a promising anti-melanogenic agent in the cosmetic industry.

The whitening effect of cuscutin responsible for traditional use of Bergenia purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and rhizomes of Bergenia purpurascens (Hook. f. et Thomson) Engl., was used as a sunscreen to protect against ultraviolet rays in Tibet of China historically, but its skin whitening constituents and pharmacological effects of this plant remained unknown. AIM OF THE STUDY: To investigate the anti-melanogenesis effect of B. purpurascens in vitro and in vivo, and then explore the preliminary mechanism. MATERIALS AND METHODS: An ultraviolet B (UVB)-induced skin injury model of mice was used to verify the ameliorative effect of B. purpurascens extract (BPE) on ultraviolet damage. Then, alpha-melanocyte stimulating hormone (α-MSH)-induced murine melanoma cell line (B16F10) melanin generation model was further adopted to approval the effects of BPE and its bioactive compound, cuscutin, in vitro. Moreover, α-MSH stimulated melanogenesis model in zebrafish was employed to confirm the anti-pigmentation effect of cuscutin. Then, proteins expressions associated with melanin production were observed using western blotting assay to explore preliminary mechanism. RESULTS: BPE inhibited UVB-induced mice injury and restored skin barrier function observably in vivo. BPE and cuscutin suppressed the overproduction of melanin in α-MSH induced B16F10 significantly, in which cuscutin exhibited better effect than well-known whitening agent α-arbutin at same 10 µg/mL concentration. Moreover, the pigmentation of zebrafish embryo was decreased by cuscutin. Finally, cuscutin showed significant downregulation of expressions of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1), TRP-2 and microphthalmia-associated transcription factor (MITF) in the melanogenic signaling pathway. CONCLUSION: B. purpurascens extract and its major bioactive constituent, cuscutin, showed potent anti-melanogenesis and skin-whitening effect by targeting TYR and TRP-2 proteins for the first time, which supported its traditional use.

Therapeutic Effects of Stimulating the Melanocortin Pathway in Regulating Ocular Inflammation and Cell Death.

Alpha-melanocyte-stimulating hormone (α-MSH) and its binding receptors (the melanocortin receptors) play important roles in maintaining ocular tissue integrity and immune homeostasis. Particularly extensive studies have demonstrated the biological functions of α-MSH in both immunoregulation and cyto-protection. This review summarizes the current knowledge of both the physiological and pathological roles of α-MSH and its receptors in the eye. We focus on recent developments in the biology of α-MSH and the relevant clinical implications in treating ocular diseases.

A comparative study on the effects of human serum albumin and α-melanocyte-stimulating hormone fusion proteins on the anti-neuroinflammatory in the central nervous system of adult mice.

The immunomodulatory effects of α-melanocyte stimulating hormone (α-MSH) in the central nervous system (CNS) have been investigated for forty years. The clinical applications of α-MSH are limited due to its short half-life. Our previous study has indicated that the short half-life of α-MSH can be extended by fusion with carrier human serum albumin (HSA) and this fusion protein has also retained the anti-inflammatory effect on the CNS. This improvement is still far from the clinical requirements. Thus, we expected to enhance the half-life and activity of the fusion protein by optimizing the linker peptide to get closer to clinical requirements. In a previous study, we screened out two candidates in vitro experiments with a flexible linker peptide (fusion protein with flexible linker peptide, FPFL) and a rigid linker peptide (fusion protein with rigid linker peptide, FPRL), respectively. However, it was not sure whether the anti-inflammatory effects in vitro could be reproduced in vivo. Our results show that FPRL is the best candidate with a longer half-life compared to the traditional flexible linker peptides. Meanwhile, the ability of FPRL to penetrate the blood-brain barrier (BBB) was enhanced, and the inhibition of TNF-α and IL-6 was improved. We also found that the toxicity of FPRL was decreased. All of the results suggested that trying to choose the rigid linker peptide in some fusion proteins may be a potential choice for improving the unsatisfactory characteristics.

Inhibition of α-melanocyte-stimulating hormone-induced melanogenesis and molecular mechanisms by polyphenol-enriched fraction of Tagetes erecta L. flower.

BACKGROUND: The pursuit for safe and efficacious skin-whitening agents has prompted a dedicated exploration of plant-derived compounds. Notably, Tagetes erecta L. flowers have been used as a medicinal extract and possessed in vitro mushroom tyrosinase activity. However, whether polyphenol-enriched fraction extracted from T. erecta L. flowers (TE) regulates melanogenesis within cellular and animal models has not yet been investigated. PURPOSE: This study aimed to investigate the effect of TE as a prospective inhibitor of melanogenesis. METHODS: Through advanced UPLC-QTof/MS analysis, the components of TE were analyzed. Anti-melanogenic effects of TE were evaluated in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells by measuring cell viability assay, extracellular and intracellular melanin biosynthesis, cyclic adenosine monophosphate (cAMP) production, and melanogenesis-related gene and protein expression. Zebrafish larvae were employed for in vivo studies, assessing both heart rate and melanogenesis. Furthermore, molecular docking analyses were employed to predict the interaction between TE components and the melanocortin 1 receptor (MC1R). Direct binding activity of TE components to MC1R was compared with [Nle4, d-Phe7]-MSH (NDP-MSH). RESULTS: TE was found to contain significant phenolic compounds such as patulitrin, quercetagetin, kaempferol, patuletin, and isorhamnetin. This study revealed that TE effectively inhibits melanin biosynthesis in both in vitro and in vivo models. This inhibition was attributed to interference of TE with the cAMP-cAMP response element-binding protein (CREB)-microphthalmia-associated transcription factor (MITF)-tyrosinase pathway, which plays a pivotal role in regulating melanogenesis. Importantly, TE exhibited the remarkable ability to curtail α-MSH-induced melanogenesis in zebrafish larvae without impacting heart rates. Molecular docking analyses predicted that the components of TE possibly interact with the melanocortin 1 receptor, suggesting their role as potential inhibitors of melanin biosynthesis. However, through the direct binding activity compared with NDP-MSH, any TE components did not directly bind to MC1R, suggesting that TE inhibits α-MSH-induced melanogenesis by inhibiting the cAMP-mediated intracellular signaling pathway. The assessment of anti-melanogenic activity, conducted both in vitro and in vivo, revealed that patulitrin and patuletin exhibited significant inhibitory effects on melanin formation, highlighting their potency as major contributors. DISCUSSION: This investigation demonstrated the considerable potential of TE as a natural remedy endowed with remarkable anti-melanogenic properties. The demonstrated capacity of TE to attenuate melanin production by modulating the cAMP-CREB-MITF-tyrosinase pathway underscores its central role in management of disorders associated with excessive pigmentation. Importantly, the implications of these findings extend to the cosmetics industry, where TE emerges as a prospective and valuable ingredient for the formulation of skin-whitening products. The elucidated interactions between TE components and MC1R not only provide insight into a potential mechanism of action but also elevate the significance of this study. In summary, this study not only contributes to our comprehension of pigmentation-related conditions but also firmly establishes TE as a secure and natural strategy for the regulation of melanin production. The innovative aspects of TE propel it into the forefront of potential interventions, marking a noteworthy advancement in the pursuit of effective and safe solutions for pigmentation disorders.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

Targeting phosphorylation circuits on CREB and CRTCs as the strategy to prevent acquired skin hyperpigmentation.

Background: The cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivators (CRTCs) cooperate in the transcriptional activation of microphthalmia-associated transcription factor subtype M (MITF-M) that is a master regulator in the biogenesis, pigmentation and transfer of melanosomes at epidermal melanocytes. Here, we propose the targeting of phosphorylation circuits on CREB and CRTCs in the expression of MITF-M as the rationale to prevent skin hyperpigmentation by elucidating the inhibitory activity and mechanism of yakuchinone A (Yaku A) on facultative melanogenesis. Methods: We employed human epidermal melanocyte cell, mouse skin, and mouse melanoma cell, and applied Western blotting, reverse transcription-polymerase chain reaction, immunoprecipitation and confocal microscopy to conduct this study. Results: This study suggested that α-melanocyte stimulating hormone (α-MSH)-induced melanogenic programs could switch on the axis of protein kinase A-salt inducible kinases (PKA-SIKs) rather than that of PKA-AMP activated protein kinase (PKA-AMPK) during the dephosphorylation of CRTCs in the expression of MITF-M. SIK inhibitors rather than AMPK inhibitors stimulated melanin production in melanocyte cultures in the absence of extracellular melanogenic stimuli, wherein SIK inhibitors increased the dephosphorylation of CRTCs but bypassed the phosphorylation of CREB for the expression of MITF-M. Treatment with Yaku A prevented ultraviolet B (UV-B)-irradiated skin hyperpigmentation in mice and inhibited melanin production in α-MSH- or SIK inhibitor-activated melanocyte cultures. Mechanistically, Yaku A suppressed the expression of MITF-M via dually targeting the i) cAMP-dependent dissociation of PKA holoenzyme at the upstream from PKA-catalyzed phosphorylation of CREB coupled with PKA-SIKs axis-mediated dephosphorylation of CRTCs in α-MSH-induced melanogenic programs, and ii) nuclear import of CRTCs after SIK inhibitor-induced dephosphorylation of CRTCs. Conclusions: Taken together, the targeting phosphorylation circuits on CREB and CRTCs in the expression of MITF-M could be a suitable strategy to prevent pigmentary disorders in the skin.

5-Hydroxypyrroloindoline Affords Tryptathionine and 2,2'-bis-Indole Peptide Staples: Application to Melanotan-II.

With peptides increasingly favored as drugs, natural product motifs, namely the tryptathionine staple, found in amatoxins and phallotoxins, and the 2,2'-bis-indole found in staurosporine represent unexplored staples for unnatural peptide macrocycles. We disclose the efficient condensation of a 5-hydroxypyrroloindoline with either a cysteine-thiol or a tryptophan-indole to form a tryptathionine or 2-2'-bis-indole staple. Judicious use of protecting groups provides for chemoselective stapling using α-MSH, which provides a basis for investigating both chemoselectivity and affinity. Both classes of stapled peptides show nanomolar Ki's, with one showing a sub-nanomolar Ki value.

Rare genetic forms of obesity in childhood and adolescence: A narrative review of the main treatment options with a focus on innovative pharmacological therapies.

The prevalence of obesity in children and adolescents is increasing, and it is recognised as a complex disorder that often begins in early childhood and persists throughout life. Both polygenic and monogenic obesity are influenced by a combination of genetic predisposition and environmental factors. Rare genetic obesity forms are caused by specific pathogenic variants in single genes that have a significant impact on weight regulation, particularly genes involved in the leptin-melanocortin pathway. Genetic testing is recommended for patients who exhibit rapid weight gain in infancy and show additional clinical features suggestive of monogenic obesity as an early identification allows for appropriate treatment, preventing the development of obesity-related complications, avoiding the failure of traditional treatment approaches. In the past, the primary recommendations for managing obesity in children and teenagers have been focused on making multiple lifestyle changes that address diet, physical activity, and behaviour, with the goal of maintaining these changes long-term. However, achieving substantial and lasting weight loss and improvements in body mass index (BMI) through lifestyle interventions alone is rare. Recently the progress made in genetic analysis has paved the way for innovative pharmacological treatments for different forms of genetic obesity. By understanding the molecular pathways that contribute to the development of obesity, it is now feasible to identify specific patients who can benefit from targeted treatments based on their unique genetic mechanisms.  Conclusion: However, additional preclinical research and studies in the paediatric population are required, both to develop more personalised prevention and therapeutic programs, particularly for the early implementation of innovative and beneficial management options, and to enable the translation of these novel therapy approaches into clinical practice. What is Known: • The prevalence of obesity in the paediatric population is increasing, and it is considered as a multifaceted condition that often begins in early childhood and persists in the adult life. Particularly, rare genetic forms of obesity are influenced by a combination of genetic predisposition and environmental factors and are caused by specific pathogenic variants in single genes showing a remarkable impact on weight regulation, particularly genes involved in the leptin-melanocortin pathway. • Patients who present with rapid weight gain in infancy and show additional clinical characteristics indicative of monogenic obesity should undergo genetic testing, which, by enabling a correct diagnosis, can prevent the development of obesity-related consequences through the identification for appropriate treatment. What is New: • In recent years, advances made in genetic analysis has made it possible to develop innovative pharmacological treatments for various forms of genetic obesity. In fact, it is now achievable to identify specific patients who can benefit from targeted treatments based on their unique genetic mechanisms by understanding the molecular pathways involved in the development of obesity. • As demonstrated over the last years, two drugs, setmelanotide and metreleptin, have been identified as potentially effective interventions in the treatment of certain rare forms of monogenic obesity caused by loss-of-function mutations in genes involved in the leptin-melanocortin pathway. Recent advancements have led to the development of novel treatments, including liraglutide, semaglutide and retatrutide, that have the potential to prevent the progression of metabolic abnormalities and improve the prognosis of individuals with these rare and severe forms of obesity. However, extensive preclinical research and, specifically, additional studies in the paediatric population are necessary to facilitate the translation of these innovative treatment techniques into clinical practice.

Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH-Peptide Conjugates.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide-drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative-daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH2). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.