Method for screening for, on basis of PTK2-ITCH-mlana-OPTN signaling cascade, substance exhibiting melanophagy inhibitory activity, and use of active substance screened for using method

Abstract

The present invention establishes a method for screening for, on the basis of a melanophagy-regulated signaling cascade involving PTK2, ITCH, MLANA and OPTN, a substance exhibiting melanophagy inhibitory activity, and provides a composition for preventing, mitigating, alleviating or treating pigmentation-related diseases, containing an active substance discovered using the developed screening method. The screening method of the present invention can effectively identify, on the basis of a novel melanophagy-regulated signaling cascade, the usefulness and potential utility of a substance exhibiting melanophagy inhibitory activity, and thus has a high utility value as a platform technology for melanophagy regulation. The composition according to the present invention has an excellent melanophagy inhibitory effect, and thus can be used in various fields such as pharmaceuticals, cosmetics and health functional foods.

Description

Method for screening substances having melanophage inhibitory activity based on the PTK2-ITCH-MLANA-OPTN signaling cascade and use of active substances screened by said method

[0001] The present invention relates to a method for screening substances having melanophage inhibitory activity based on signal transduction mechanisms involved in the regulation of melanophages. Furthermore, the present invention relates to using effective substances discovered through a newly established screening method to prevent, alleviate, improve, or treat pigmentation-related diseases.

[0002] Cell organelles are complex entities within a cell that perform specific functions important for cell function and survival. These highly specialized structures are precisely regulated through meticulous biogeneration and degradation processes based on environmental and functional needs, ensuring the equilibrium and adaptability of cellular life [1,2]. Melanosomes are pivotal organelles of this complex biological system that act as designers of skin color and photoprotection [3]. They facilitate numerous cellular processes, one of the most important of which is melanogenesis. This is a highly regulated and multifaceted process involving the synthesis, translocation, and release of melanin, the pigment responsible for the color of the skin, hair, and eyes [4,5].

[0003] Alpha-melanocyte-stimulating hormone (α-MSH) is important for stimulating melanin production. When α-MSH binds to the melanocortin 1 receptor (MC1R), a receptor on the surface of melanocytes, it activates intracellular signaling pathways that induce melanin production and release [6-8]. Melanin production is primarily controlled by MITF (microphthalmia-associated transcription factor). MITF carefully regulates the expression of several proteins essential for melanin synthesis, including tyrosinase, tyrosinase-associated protein 1 (TYRP-1), and TYRP-2 [9,10]. The regulation of melanin production is important due to its therapeutic potential, particularly when designing interventions for skin hyperpigmentation by targeting the enzymatic activity of melanogenic proteins [5].

[0004] Macroautophagy, also known as autophagy, is a conserved lysosome-dependent degradation pathway essential for maintaining cellular homeostasis

[0011] . Autophagy is characterized by the selective degradation of cellular components with specific cargoes that are regulated by E3 ligases, labeled by ubiquitin, and subsequently recognized by autophagy adapter proteins such as sequestosome 1 (SQSTM1 / p62) [12,13]. Among the various types of selective autophagy, mitophagy is a significantly prominent specialty of autophagy that focuses on mitochondrial degradation by autophagy

[0014] . Mitophagy is a well-organized process in which PTEN-induced kinase 1 (PINK1) accumulates in damaged mitochondria and mobilizes the E3 ligase Parkin. This PINK1-Parkin axis is important for mediating the ubiquitination of mitochondrial outer membrane proteins, including voltage-dependent anion channel 1 (VDAC1) and mitofusin [15-19]. Ubiquitinated mitochondrial outer membrane proteins are recognized by autophagy adapter proteins such as SQSTM1 and Optineurin (OPTN), which trigger the recruitment of autophagy machinery to degrade damaged mitochondria [17,20]. The integrity of the PINK1-Parkin-VDAC1-SQSTM1 scenario, a mitophagy pathway, is important for maintaining cell health and preventing the accumulation of dysfunctional mitochondria.

[0005] While melanogenesis is important for maintaining melanosome homeostasis by regulating melanin synthesis, melanophages, a specialized form of autophagy for melanosomes, are important for the degradation of melanosomes [21,22]. This unique interaction between melanogenesis and melanophages is key to maintaining intracellular melanin balance and represents a fine interconnection between production and degradation pathways that determine cellular melanin levels. Recent studies have highlighted the concept of melanophages by discovering a link between autophagy and melanogenesis in melanocytes and keratinocytes [24,25]. In particular, 3,4,5-trimethoxycinnamate thymol ester (TCTE, also known as Melasolv™) acts as a potent inhibitor of skin pigmentation by downregulating key pigment cell-specific enzymes such as tyrosinase, TYRP-1 / 2, and MITF, which are important in melanogenesis [26-28]. Interestingly, the inventors have demonstrated that TCTE treatment enhances autophagy and reduces melanin content

[0029] . Since the role of autophagy in melanin degradation has a significant impact on skin pigmentation, this convergence is important for understanding skin color changes

[0030] .

[0006] Although previous research has broadened our understanding of melanophages, there are still many limitations in precisely controlling their mechanisms; consequently, there is still a need for in-depth research into their regulatory mechanisms.

[0007] The inventors have made diligent efforts to elucidate the complex mechanisms and subtle nuances of melanosome-selective autophagy using a specially designed advanced monitoring system. As a result, the inventors discovered a new signaling cascade involved in melanophage regulation, namely the PTK2 (Protein Tyrosine Kinase 2)-ITCH (Itchy E3 ubiqutin ligase)-MLANA (Melan-A)-OPTN (Optineurin) signaling cascade, and confirmed that ITCH plays a pivotal role. Furthermore, the inventors confirmed that melanophages can be regulated through the PTK2-ITCH-MLANA-OPTN signaling cascade in B16F1 cells treated with melanophage-inducing substances such as TCTE, and that this can be utilized as an innovative approach to address skin pigmentation problems, thereby completing the present invention.

[0008] This invention was made possible through the support of the Korea Health Technology R&D Project of the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare (Project No.: HP24C1234).

[0009]

[0010] The present invention aims to identify a novel signaling mechanism involved in the regulation of melanophages and to establish a screening method for substances having melanophage inhibitory activity using this.

[0011] The present invention aims to use a substance determined to be an effective substance through an established screening method to prevent, alleviate, improve, or treat pigmentation-related diseases.

[0012] To achieve the aforementioned objective, the present invention provides a method for screening substances having melanophage inhibitory activity based on the PTK2-ITCH-MLANA-OPTN signaling cascade.

[0013] In addition, the present invention provides a pharmaceutical use of 2,3-dichloro-1,4-naphthoquinone (also referred to as 'dichlone' in this specification) or 1,2,4,5-benzenetetraamine tetrahydrochloride (also referred to as 'Y15' in this specification), which is determined to be an active substance according to the screening method described above. Specifically, the present invention provides a pharmaceutical composition for use in preventing, alleviating, or treating hypopigmentation-related diseases, comprising 2,3-dichloro-1,4-naphthoquinone (dichlone), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0014] In addition, the present invention provides a use of a diclon determined to be an active substance according to the screening method described above as a topical skin composition. Specifically, the present invention provides a topical skin composition for use in preventing, alleviating, or improving hypopigmentation-related diseases, comprising 2,3-dichloro-1,4-naphthoquinone (diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0015] In addition, the present invention provides a health functional food use of a diclon determined to be an active substance according to the screening method described above. Specifically, the present invention provides a health functional food composition for use in preventing, alleviating, or improving hypopigmentation-related diseases, comprising 2,3-dichloro-1,4-naphthoquinone (diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0016]

[0017] The present invention can easily determine the efficacy and utility of substances having melanophage inhibitory activity based on the PTK2-ITCH-MLANA-OPTN cascade, a newly identified melanophage regulatory signaling pathway. The present invention can provide substances with excellent effects in preventing, alleviating, improving, or treating hypopigmentation-related diseases. By providing an in-depth understanding of melanophage-related abnormalities or disorders, the present invention can present a new approach to developing innovative therapies and cosmetic strategies to address the problem of hypopigmentation.

[0018] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It should be noted, by general practice, that various features in the drawings do not correspond to their actual dimensions. Rather, the dimensions of various features have been arbitrarily enlarged or reduced for clarity. The following drawings are attached, and the features of the present invention and their equivalents will be clearly understood by those skilled in the art by referring to the following description with reference to the attached drawings:

[0019] Figures 1a-1e: Figures showing the involvement of ITCH in TCTE-induced melanophages in B16F1 cells. (a) B16F1 cells stimulated with α-MSH were transfected with EGFP-TYR and mRFP-UBC. Cells were cotreated with α-MSH (1 μM) and TCTE (10 μg / ml) for 24 hours. Then, cells were fixed and imaged using a confocal microscope. Co-localization of TYR and UBC was calculated using Pearson correlation coefficients. Data are presented as mean ± SEM (n = 6, * p < 0.001). The scale bar represents 10 μm. (b and c) B16F1 cells stably expressing TPC2-mRFP-EGFP (B16F1 / TPC2-mRFP-EGFP) were transfected with scrambled siRNA (Sc) or Atg5-targeted siRNA (siAtg5). After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were fixed, and only the number of mRFP spots per cell was counted using merged images. Data are presented as mean ± SEM (n = 50, * p < 0.001). The scale bar represents 10 μm. (c) Knockdown efficiency was evaluated by Western blotting using the indicated antibodies. (d) B16F1 / TPC2-mRFP-EGFP cells stimulated with α-MSH were treated with TCTE (10 μg / ml) for 24 hours in the presence or absence of dichlone (DC, 25 μM). Then, the cells were fixed and imaged using a confocal microscope. Only the number of mRFP spots per cell was counted from the merged images. Data are presented as mean ± SEM (n = 15, * p < 0.001). The scale bar represents 10 μm. (e) B16F1 / TPC2-mRFP-EGFP cells were transfected with scrambled siRNA (Sc) or Itch-targeting siRNA (siItch).After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were fixed, and only the number of mRFP spots was counted using merged images. Data are presented as mean ± SEM (n = 50, * p < 0.001). The scale bar represents 10 μm.

[0020] Figures 2a-2h: Figures showing that MLANA (Melan-A) is ubiquitinated by ITCH and interacts with OPTN in TCTE-induced melanophages. (a) B16F1 / TPC2-mRFP-EGFP cells were transfected with scrambled siRNA (Sc) or Mlana-targeting siRNA (siMlana). After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were fixed, and only the number of mRFP spots per cell was counted using merged images. Data are presented as mean ± SEM (n = 50, * p < 0.001). The scale bar represents 10 μm. (b) B16F1 cells stimulated with α-MSH were transfected with Myc-Itch along with Flag or MLANA-Flag. After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and then with TCTE (10 μg / ml) along with bafilomycin A1 (5 nM) for an additional 24 hours. Then, cells were treated with MG132 (10 μM) for an additional 4 hours and precipitated with anti-Flag antibodies conjugated to agarose beads. Immunoprecipitates were analyzed by Western blotting with the labeled antibodies. (c) B16F1 cells stimulated with α-MSH were transfected with MLANA-Flag and EGFP-UBC. Cells were co-treated with α-MSH (1 μM) and TCTE (10 μg / ml) for 24 hours. Then, cells were fixed, stained with anti-Flag antibodies, and images were obtained using a confocal microscope. Colocalization of Flag and UBC was estimated using Pearson's correlation coefficient. Data are presented as mean ± SEM (n = 6, * p < 0.001). Scale bars represent 10 μm. (d) B16F1 cells stimulated with α-MSH were transfected with Flag or MLANA-Flag for 24 hours.Then, cells were treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) with bafilomycin A1 (5 nM) for an additional 24 hours. Then, cells were treated with MG132 (10 μM) for an additional 4 hours and precipitated with an anti-Flag antibody conjugated to agarose beads. The immunoprecipitate was analyzed by Western blotting using the labeled antibody. (e) In vitro ubiquitination assay of MLANA by ITCH. HEK293T cells were transfected with Myc or Myc-Itch for 24 hours. Then, cells were precipitated using an anti-Myc antibody conjugated to agarose beads. The immunoprecipitate was incubated with purified MLANA protein for 2 hours and then analyzed by Western blotting using the labeled antibody. (f) B16F1 cells stimulated with α-MSH were transfected with scrambled siRNA or Itch-targeting siRNA (siItch) in combination with HA-UBC and MLANA-Flag. After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and further exposed to TCTE (10 μg / ml) with bafilomycin A1 (5 nM) for 24 hours. Then, cells were further treated with MG132 (10 μM) for 4 hours and precipitated with anti-Flag antibodies conjugated to agarose beads. Immunoprecipitates were analyzed by Western blotting with the labeled antibodies. (g) B16F1 / TPC2-mRFP-EGFP cells were transfected with scrambled siRNA (Sc) or Optn-targeting siRNA (siOptn). After 1 day, cells were treated with α-MSH (1 μM) for an additional 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Cells were fixed, and only the number of mRFP spots per cell was counted using merged images. Data are presented as mean ± SEM (n = 50, * p < 0.001). The scale bar represents 10 μm.(h) B16F1 cells stimulated with α-MSH were transfected with MLANA-Flag along with EGFP or EGFP-OPTN. After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) with bafilomycin A1 (5 nM) for an additional 24 hours. Then, cells were treated with MG132 (10 μM) for an additional 4 hours and precipitated with anti-Flag antibodies conjugated to agarose beads. Immunoprecipitates were analyzed by Western blotting using the labeled antibodies.

[0021] Figures 3a-3h: Figures showing that when PTK2 is activated in response to TCTE, ITCH is phosphorylated, promoting melanophage in B16F1 cells. (a) B16F1 cells stimulated with α-MSH were treated with TCTE (10 μg / ml) for 24 hours, and Proteome Profiler Human Phosphor-Kinase array analysis was performed. Highlighted spots indicate PTK2 phosphorylated at Y397, and relative intensity was measured by densitometry. (B) B16F1 cells stimulated with α-MSH were co-treated with TCTE (10 μg / ml) and the PTK2 inhibitor Y15 (1.5 μM) for 24 hours. Then, cells were harvested and analyzed by Western blotting using the labeled antibodies. (c) α-MSH-stimulated B16F1 / TPC2-mRFP-EGFP cells were co-treated with TCTE (10 μg / ml) and Y15 (1.5 μM) for 24 hours. Then, the cells were fixed and imaged using a confocal microscope. Only the number of mRFP spots per cell was counted from the merged images. Data are presented as mean ± SEM (n = 10, * p < 0.001). The scale bar represents 10 μm. (d) α-MSH-stimulated B16F1 cells were co-transfected with GFP-Ptk2 and Myc-Itch. After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and further exposed to TCTE (10 μg / ml) containing bafilomycin A1 (5 nM) for 24 hours. Then, cells were additionally treated with MG132 (10 μM) for 4 hours and precipitated with anti-Myc antibodies conjugated to agarose beads. The immunoprecipitates were analyzed by Western blotting using the labeled antibodies. (e) B16F1 cells stimulated with α-MSH were transfected with Myc-Itch for 24 hours.Cells were further treated with α-MSH (1 μM), TCTE (10 μg / ml), and bafilomycin A1 (5 nM) for 24 hours. Then, cells were further treated with MG132 (10 μM) for 4 hours and precipitated with anti-phosphorusserin antibodies conjugated to agarose beads. Immunoprecipitates were analyzed by Western blotting using the labeled antibodies. (f) B16F1 cells stimulated with α-MSH were co-transfected with HA-UBC and MLANA-Flag. After 1 day, cells were further treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) with Y15 (1.5 μM) or DC (25 μM) for 24 hours. Then, cells were treated with MG132 (10 μM) for an additional 4 hours and precipitated with an anti-Flag antibody conjugated to agarose beads. The immunoprecipitate was analyzed by Western blotting using the labeled antibody. (g) B16F1 cells stimulated with α-MSH were co-transfected with MLANA-Flag and OPTN-GFP for 24 hours. After 1 day, cells were co-treated with α-MSH (1 μM), TCTE (10 μg / ml), and bafilomycin A1 (5 nM) for 24 hours, along with Diclon (25 μM, DC) and Y15 (1.5 μM, Y15). Then, cells were treated with MG132 (10 μM) for an additional 4 hours and precipitated using an anti-Flag antibody conjugated to agarose beads. The immunoprecipitate was analyzed by Western blotting using the labeled antibody. (h) Schematic diagram of a novel melanophage mechanism associated with the PTK2-ITCH-MLANA-OPTN cascade. TCTE exposure prevents melanosomes from completing maturation. As a sensor for abnormal melanosomes, the tyrosine kinase PTK2 is activated by Y397 phosphorylation. Activated PTK2 promotes the phosphorylation of the HECT-type family E3 ligase ITCH, and active ITCH promotes the ubiquitination of the melanosomal membrane protein MLANA.Ubiquitinated MLANA is recognized by the autophagy adapter OPTN, leading to melanosome autophagy degradation.

[0022] Figure 4: This figure illustrates the co-localization of LC3 and tyrosinase in TCTE-treated B16F1 cells. B16F1 cells stimulated with α-MSH were transfected with EGFP-TYR and mRFP-LC3. Cells were co-treated with α-MSH (1 μM) and TCTE (10 μg / ml) for 24 hours. Subsequently, the cells were fixed and imaged using a confocal microscope. The co-localization of TYR and LC3 was calculated using Pearson correlation coefficients. Data are presented as mean ± SEM (n = 10, * p < 0.001). The scale bar represents 10 μm.

[0023] Figure 5: A figure showing the screening results of various melanophage inhibitors. Determination of the relative grades of melanophage inhibitors through ubiquitination compound library screening in B16F1 / TPC2-mRFP-EGFP cells treated with TCTE. Bafilomycin A1 (10 nM, Baf.A1) was treated as a positive control for melanophage inhibitors. The proportion of cells exhibiting melanophages was evaluated and classified into grades 1 to 5. Grade 1 indicates less than 10% melanophages (e.g., bafilomycin A1 and TCTE), Grade 2 indicates 10 to 30%, Grade 3 indicates 30 to 60%, Grade 4 indicates 60 to 80%, and Grade 5 indicates more than 80% (e.g., TCTE alone). Data are presented as mean ± SEM (n = 2).

[0024] Figure 6: This figure shows the screening results for melanosomal membrane proteins. B16F1 / TPC2-mRFP-EGFP cells were transfected with scrambled siRNA (Sc) or siRNA targeting melanosomal membrane proteins containing siMlana. After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were fixed, and the number of mRFP spots per cell was counted using merged images. Data are presented as mean ± SEM (n = 8, *** p < 0.001, ** p < 0.01, ** p < 0.05). The scale bar represents 10 μm.

[0025] Figure 7: This figure shows the screening results for melanophage adapter proteins. B16F1 / TPC2-mRFP-EGFP cells were transfected with melanophage adapter protein-targeting siRNA containing scrambled siRNA (Sc) or siOptn. After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were fixed, and the number of mRFP spots per cell was counted using merged images. Data are presented as mean ± SEM (n = 8, * p < 0.001). The scale bar represents 10 μm.

[0026] Figure 8: A figure showing the knockdown efficiency and the effect of siRNA sequences on melanophage flux. B16F1 / TPC2-mRFP-EGFP cells were transfected with scrambled siRNA (Sc) or melanophage regulator-targeting siRNAs such as siItch (A), siMlana (B), and siOptn (C). After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were harvested and knockdown efficiency and melanophage flux were analyzed by Western blotting using the labeled antibodies.

[0027] Figure 9: This figure shows that the loss of ITCH and OPTN inhibits the depigmentation activity of TCTE in B16F1 cells, whereas MLANA does not. B16F1 cells were transfected with scrambled siRNA (Sc) or siRNAs targeting melanophage regulators such as siItch (A), siMlana (B), and siOptn (C). After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) for an additional 24 hours. Then, cells were harvested and melanin content was analyzed. Data are presented as mean ± SEM (n = 4, ** p < 0.01, * p < 0.05). NS indicates non-specific.

[0028] Fig. 10: This figure shows that inhibition of ITCH or PTK2 reduces melanophage flux and MLANA ubiquitination in Melan-A cells treated with TCTE. (A) Melan-A cells stimulated with α-MSH were transfected with TPC2-mRFP-EGFP for 48 hours and treated with TCTE (10 μg / ml) along with Diclon (DC, 25 μM) or the PTK2 inhibitor Y15 (1.5 μM) for an additional 24 hours. The cells were then fixed and imaged using a confocal microscope. Only the number of mRFP spots per cell was counted from the merged images. Data are presented as mean ± SEM (n = 40, * p < 0.001). The scale bar represents 10 μm. (B) Melan-A cells stimulated with α-MSH were co-transfected with MLANA-Flag and HA-UBC. After 1 day, cells were additionally treated with α-MSH (1 μM) for 48 hours and exposed to TCTE (10 μg / ml) with Y15 (1.5 μM) or DC (25 μM) for 24 hours. Then, cells were additionally treated with MG132 (10 μM) for 4 hours and precipitated with an anti-Flag antibody conjugated to agarose beads. The immunoprecipitate was analyzed by Western blotting using the labeled antibody.

[0029] definition

[0030] Before describing exemplary embodiments in more detail, the following definitions are provided to explain and define the meaning and scope of the terms used in the detailed description.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. The literature [Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991)] provides a person skilled in the art with the general meaning of many terms used herein. However, for clarity and ease of reference, specific terms are defined below.

[0032] Specific ranges are presented in this specification along with numerical values ​​preceded by the term “about.” The term “about” is used in this specification to provide literal support for numbers that are close to or approximate the number that follows it, as well as for the exact number that follows it. When determining whether a number is close to or approximate a specifically mentioned number, a number that is close to or approximate an unmentioned number may be a number that provides a substantially equivalent to the specifically mentioned number in the context in which it is presented.

[0033] It should be noted that the singular form used in this specification and the appended claims includes the plural form unless the context clearly indicates otherwise.

[0034] As used herein, the term “polynucleotide” may be used interchangeably with “nucleic acid” and refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxynucleotides. Accordingly, the term includes, but is not limited to, polymers comprising single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides where applicable to the described embodiments.

[0035] The term “protein” as used herein may be used interchangeably with “peptide” and “polypeptide” and refers to a polymer of amino acids of any length, which may include coded amino acids and uncoded amino acids, amino acids that have been chemically or biochemically modified or derivatized, and polypeptides having a modified peptide backbone.

[0036] As used herein, the term “transfection” may be used interchangeably with “transformation” and refers to a permanent or transient genetic change induced in a cell following the introduction of nucleic acids (i.e., DNA and / or RNA that are exogenous to the cell). Genetic change (“modification”) can be achieved by incorporating new DNA into the host cell’s genome or by maintaining new DNA transiently or stably as an episomal element. If the cell is a eukaryotic cell, permanent genetic change is generally achieved by introducing DNA into the cell’s genome. Suitable methods for genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, and direct microinjection. The choice of method generally depends on the type of cell being transformed and the circumstances under which the transformation occurs (i.e., in vitro, ex vivo, or in vivo). General discussions on these methods can be found in the literature [Ausubel et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995].

[0037] As used herein, the term “operably linked” means being in a relationship that allows the described components to function in an intended manner. For example, if a promoter influences the transcription and / or expression of any polynucleotide, the promoter is operably linked to a nucleotide sequence (e.g., a protein-coding sequence, e.g., a sequence coding for mRNA; a non-protein-coding sequence, e.g., a sequence coding for Shh protein, etc.).

[0038] As used herein, the term "melanophage" refers to the breakdown of melanosomes present in skin cells through autophagy.

[0039] As used herein, the term "melanosome" is used interchangeably with "melanosomes" and refers to a cellular organelle containing melanin, the most common light-absorbing pigment found in the animal kingdom. Cells that produce melanosomes are referred to as melanocytes, and cells that phagocytose melanosomes are referred to as melanophagocytes.

[0040] details

[0041] Before describing various embodiments, it should be understood that the teachings of the present invention are not limited to the specific embodiments described and may vary. Furthermore, since the scope of these teachings is limited only by the appended claims, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0042] Section headings used in this specification are for listing purposes only and should not be interpreted as limiting the subject matter described in any way. Although the invention is described with various embodiments, it is not intended to be limited to such embodiments. Rather, the invention includes various alternatives, modifications, and equivalents as would be understood by a person skilled in the art.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this disclosure pertains. Any method and material similar or equivalent to that described herein may be used in the practice or testing of this teaching, but some exemplary methods and materials are described below.

[0044] References to any publications are for disclosure prior to the filing date and should not be construed as an acknowledgment that the claims are not entitled to precede such publications due to prior art. Additionally, the provided disclosure date may differ from an actual disclosure date that can be independently verified.

[0045] As will be apparent to a person skilled in the art when reading the invention, each individual embodiment described and illustrated herein has distinct components and features that can be easily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any mentioned method may be performed in the order of the mentioned events or in any other logically possible order.

[0046] All patents and publications containing all sequences disclosed in the patents and publications mentioned in this specification are incorporated by reference herein by reference.

[0047] Where a range of values ​​is provided, each intermediate value is included in the invention up to one-tenth of a unit of the lower limit, between the upper and lower limits of the range and other specified or intermediate values ​​within the range, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the invention according to any limits specifically excluded from the mentioned ranges. Where the specified range includes one or both of the limits, the range excluding either or both of the included limits is also included in the invention.

[0048] For clarity, it is understood that specific features of the invention described in relation to separate embodiments may be provided in combination with a single embodiment. Conversely, for simplification, various features of the invention described in the context of a single embodiment may be provided separately or in any suitable sub-combination. All combinations of embodiments falling under the invention are specifically incorporated into the invention and are disclosed herein as if each and all combinations were individually and explicitly disclosed. Furthermore, all sub-combinations of various embodiments and elements thereof are also specifically incorporated into the invention and are disclosed herein as if each and all sub-combinations were individually and explicitly disclosed.

[0049] In addition, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the invention described in this application using only ordinary experiments. In addition, such equivalents are intended to be included in the invention.

[0050] Screening method for substances having melanophage inhibitory activity

[0051] As a specific embodiment of the present invention, a screening method for a substance having melanophage inhibitory activity based on the PTK2-ITCH-MLANA-OPTN signaling cascade is provided, the method comprising: a. preparing B16F1 melanoma cells (abbreviated herein as 'B16F1 / TPC2-mRFP-EGFP cells') into which an expression cassette comprising mRFP (monomeric Red Fluorescent Protein) and EGFP (Enhanced Green Fluorescent Protein) operably linked to TPC2 (Two pore segment channel 2) has been introduced; b. inducing melanophages in the prepared B16F1 melanoma cells by treating them with a substance that induces melanophages, either without a test substance or with a test substance, and stimulating the cells by treating them with alpha-melanocyte-stimulating hormone (α-MSH); c. The method is characterized by comprising the steps of: fixing the cells, capturing a fluorescence image of the cells to generate an image combining the red fluorescence of mRFP and the green fluorescence of EGFP, and measuring the number of puntas exhibiting red fluorescence from the generated image; d. determining the test substance that reduces the number of puntas exhibiting red fluorescence compared to the case where TCTE is treated without the test substance as an effective substance having melanophage inhibitory activity.

[0052] In the present invention, the melanophage-inducing substance may be a substance that exhibits whitening activity through the melanophage pathway, and a representative example is 3,4,5-trimethoxycinnamate thymol ester (TCTE).

[0053] In the present invention, B16F1 melanoma cells may be constructed by subcloning the polynucleotide encoding TPC2 and the polynucleotide encoding mRFP-EGFP, respectively, into a suitable mammalian expression plasmid such as pcDNA™ 3.1, then ligating each plasmid to produce a plasmid that stably expresses TPC2-mRFP-EGFP, and transfecting with this plasmid. As long as TPC2-mRFP-EGFP can be stably expressed, there are no particular restrictions on the plasmid used and the method of construction.

[0054] In the present invention, a melanophage-inducing substance such as TCTE can induce melanophages in B16F1 melanoma cells through the PTK2-ITCH-MLANA-OPTN signaling cascade, which consists of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment.

[0055] The treatment concentration of a melanophage-inducing substance such as TCTE may be changed as needed, but to induce melanophages while maintaining the viability of B16F1 melanoma cells, it may be 5 to 15 μg / ml, preferably 8 to 12 μg / ml, more preferably 10 μg / ml.

[0056] In the present invention, the term “substance under test” refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The substance under test includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0057] In the present invention, the test substance may be treated substantially simultaneously with or after treatment with a melanophage-inducing substance such as TCTE. The treatment concentration of the substance under test may vary depending on the substance, but is approximately 0.1 to approximately 250 μM, approximately 0.5 to approximately 240 μM, approximately 1 to approximately 230 μM, approximately 1.5 to approximately 220 μM, approximately 2 to approximately 210 μM, approximately 2.5 to approximately 200 μM, approximately 3 to approximately 190 μM, approximately 3.5 to approximately 180 μM, approximately 4 to approximately 170 μM, approximately 4.5 to approximately 160 μM, approximately 5 to approximately 150 μM, approximately 5.5 to approximately 140 μM, approximately 6 to approximately 130 μM, approximately 6.5 to approximately 120 μM, approximately 7 to approximately 110 μM, approximately 7.5 to approximately 100 μM, approximately 8 to approximately 90 μM, and approximately 8.5 to approximately 80 It may be μM, about 9 to 70 μM, about 9.5 to about 60 μM, about 10 to about 50 μM, about 20 to about 40 μM, or a sub-range within the aforementioned range.

[0058] In the present invention, the total treatment time of the test substance and the melanophage-inducing substance such as TCTE may be changed as needed, but may be about 20 to about 28 hours, preferably about 22 to about 26 hours, more preferably about 24 hours, in order to induce an appropriate response while maintaining the survival of B16F1 melanoma cells.

[0059] In the present invention, α-MSH is administered to stimulate cell proliferation and pigmentation by amplifying intracellular cAMP. α-MSH may be administered substantially simultaneously with or after the administration of a melanophage-inducing substance such as TCTE. The treatment concentration of α-MSH may be about 0.5 to about 1.5 μM, preferably about 0.8 to about 1.2 μM, more preferably about 1.0 μM, although it may be varied as needed. α-MSH may be administered for about 22 to about 50 hours, preferably about 24 to about 48 hours, more preferably about 24 to about 36 hours, to induce appropriate cell proliferation and melanin pigmentation, although this may be varied as needed.

[0060] In the present invention, B16F1 / TPC2-mRFP-EGFP cells can be fixed using conventional reagents such as paraformaldehyde, but any reagent and method may be applied on the premise that it does not affect the detection of fluorescence resulting from the expression of fluorescent proteins from the cells.

[0061] In the present invention, fluorescence can be confirmed by irradiating a cell with light of a wavelength of 400 to 700 nm, for example, 485 nm to 700 nm, 485 nm to 650 nm, 485 nm to 600 nm, 485 nm to 590 nm, 485 nm to 580 nm, or 485 nm to 570 nm, although this is not limited thereto.

[0062] In the present invention, a fluorescence image of a cell is obtained by irradiating the cell with light of an appropriate wavelength and then capturing the EGFP fluorescence and mRFP fluorescence appearing in the cell using a confocal microscope, more specifically a confocal fluorescence microscope, and a combined image of EGFP and mRFP can be obtained using an image processing program such as ImageJ(NIH).

[0063] The inventors confirmed that in B16F1 / TPC2-mRFP-EGFP cells treated with a melanophage-inducing substance such as TCTE, green fluorescence decreased and red fluorescence significantly increased as melanophage was induced, and discovered that the melanophage-inhibiting activity of the substance can be determined by measuring (counting) the number of puntas showing red fluorescence from the combined image of EGFP and mRFP.

[0064] According to the screening method of the present invention, the melanophage inhibitory activity of a test substance can be determined within a short time of about 48 to 50 hours, typically about 24 to 36 hours, through simple fluorescence measurement, so it is particularly useful.

[0065] In the present invention, if the number of spots exhibiting red fluorescence when a melanophage-inducing substance such as TCTE is treated together with a test substance is reduced by at least 30%, preferably at least 40%, and more preferably at least 50% compared to the number of spots exhibiting red fluorescence when a melanophage-inducing substance such as TCTE is treated without a test substance, the substance can be determined to have effective melanophage inhibitory activity.

[0066] In the present invention, since melanophages are fundamentally based on the PTK2-ITCH-MLANA-OPTN signaling cascade, the significance of the determination result can be further verified by additionally confirming whether the test substance determined to be an effective substance weakens or blocks at least one signaling pathway of the PTK2-ITCH-MLANA-OPTN signaling cascade, which consists of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment.

[0067] The expression and / or activity levels of PTK2, ITCH, MLANA (Melan-A), and OPTN (Optineurin) may be measured using one or more selected from the group consisting of reverse transcription-polymerase chain reaction (RTPCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting, and flow cytometry (FACS), but are not limited thereto.

[0068] In particular, it should be noted that the screening method according to the present invention can discover test substances that exhibit activity of inhibiting ITCH (Itchy E3 ubiqutin ligase) activity, inhibiting the phosphorylation of ITCH, or weakening or inhibiting the interaction between ITCH and MLANA (Melan-A).

[0069] Since the screening method according to the present invention induces melanophages through treatment with a melanophage-inducing substance exhibiting whitening activity, it may also verify whether a melanophage-inducing substance such as TCTE treated in step b) (including substances currently known to exhibit whitening activity through the melanophage pathway as well as any substances that may be found to exhibit such whitening activity in the future) exhibits whitening activity through the PTK2-ITCH-MLANA-OPTN signaling cascade pathway, which consists of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment.

[0070] Examples of active substances discovered through the screening method according to the present invention include 2,3-dichloro-1,4-naphthoquinone (diclon) or 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15).

[0071] The screening method according to the present invention is based on B16F1 / TPC2-mRFP-EGFP cells that stably express TPC2-mRFP-EGFP, so it has high reliability and allows the entire process to be easily performed. Furthermore, since the activity of a test substance can be analyzed by measuring only the number of fluorescent spots within a relatively short time, it has high utility as a platform technology for various substances.

[0072] Composition and Uses

[0073] As previously discussed, through the screening method according to the present invention, it was confirmed that 2,3-dichloro-1,4-naphthoquinone (diclon) and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) exhibit melanophage inhibitory activity.

[0074] In another embodiment of the present invention, a pharmaceutical composition for use in preventing, alleviating, or treating hypopigmentation-related diseases is provided, comprising 2,3-dichloro-1,4-naphthoquinone (diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0075] In the present invention, the hypopigmentation-related disease may be any one selected from the group consisting of, but is not limited to, vitiligo, albinism, partial albinism, depigmented nevus, chemical-induced white skin disease, idiopathic guttate hypomelanosis, punctate hypomelanosis, Freddy-Willi syndrome, Waddenbrook syndrome, Vogt-Koyanagi-Harada syndrome, fungal infection (tinea versicolor), pityriasis whiteis (ringworm), and Ito hypomelanosis.

[0076] As used herein, the term "prevention" means preventing, in the short or long term, the occurrence of abnormal symptoms resulting from the abnormal accumulation of pigment in the body or organs (e.g., reduced pigmentation), including diseases related to hypopigmentation in cells or organs (e.g., the eye), by means of the pharmaceutical composition of the present invention.

[0077] As used herein, the term "alleviation" means weakening or reducing hypopigmentation-related diseases and related symptoms occurring in cells or organs (e.g., the eye) by means of the pharmaceutical composition of the present invention.

[0078] As used herein, the term "treatment" refers to any phenomenon in which abnormal symptoms, disorders, or disease states resulting from the abnormal accumulation or deposition of pigment in cells or organs (e.g., the eye) are improved or beneficially altered by the pharmaceutical composition of the present invention.

[0079] In the present invention, the content of 2,3-dichloro-1,4-naphthoquinone (Diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof included in the pharmaceutical composition can be appropriately adjusted according to the method of use, the condition of the user, and the type and severity of the disease.

[0080] In the present invention, the pharmaceutical composition may additionally include an ingredient effective for treating, improving, alleviating, reducing, or delaying diseases related to hypopigmentation or related symptoms caused by such diseases.

[0081] In the present invention, the pharmaceutical composition may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include, but are not limited to, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, etc. The pharmaceutically acceptable additive according to the present invention may be included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0082] In the present invention, pharmaceutical compositions may be formulated into various formulations. When formulating, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents, excipients, or mixtures thereof may be used. The document [Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006], the full text of which is incorporated herein by reference, discloses various excipients used to formulate pharmaceutical compositions and known techniques for manufacturing them.

[0083] Carriers, excipients, and diluents that may be included in the pharmaceutical composition include, but are not limited to, lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0084] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium styrate talc may be used in addition to simple excipients. Furthermore, liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0085] Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, Macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc., may be used as bases for suppositories. Parenteral administration may be performed using topical application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0086] In a specific embodiment of the present invention, a method for preventing, alleviating, or treating a pigmentation-related disease is provided, comprising the step of administering a pharmaceutical composition according to the present invention to a subject.

[0087] The terms "pharmaceutical composition," "hypopigmentation-related disease," "prevention," and "treatment" are as described above.

[0088] The term "subject" refers to all animals, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, that have developed or may develop hypopigmentation-related diseases, and the said abnormalities or disorders can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention may be administered in conjunction with existing therapeutic agents.

[0089] As used herein, the term "administration" means providing a specific substance to a subject by any appropriate method.

[0090] The administration route of the pharmaceutical composition of the present invention may be any general route capable of reaching the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto. Additionally, the pharmaceutical composition of the present invention may be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectable preparations can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin), etc., and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0091] Administration may be performed in a pharmaceutically effective amount. As used herein, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0092] For example, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0093] Specifically, the amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and body weight, and generally, 1 to 100 mg, preferably 5 to 60 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the dosage and range presented in this specification do not limit the scope of the present invention in any way.

[0094] In the present invention, the pharmaceutical composition may be used alone or in combination with methods such as surgery, radiation therapy, hormone therapy, chemotherapy, and the use of biological response modifiers for the prevention, alleviation, or treatment of hypopigmentation-related diseases.

[0095] In another embodiment of the present invention, a topical skin composition for use in preventing, alleviating, or improving hypopigmentation-related diseases is provided, comprising 2,3-dichloro-1,4-naphthoquinone (Diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0096] The terms "hypopigmentation-related disease," "prevention," and "alleviation" are as described above.

[0097] As used herein, the term "improvement" may be used interchangeably with the aforementioned "alleviation" or "treatment," and refers to any phenomenon in which abnormal symptoms, disorders, or disease states related to hypopigmentation occurring in cells or organs (e.g., the eye) are improved or beneficially altered by the topical skin composition of the present invention.

[0098] The external skin composition according to the present invention can be formulated into a quasi-drug or cosmetic.

[0099] In the present invention, the quasi-drug composition can perform the function of normalizing the amount of melanin pigment deposited on the skin or helping to restore the original skin color, and can prevent abnormal deposition of pigment on the skin by inhibiting melanophages and inhibiting the degradation of melanosomes.

[0100] The quasi-drug composition of the present invention may further include, in addition to the above components, a pharmaceutically acceptable carrier, excipient, or diluent as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impede the effects of the present invention, and may include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc.

[0101] The composition of the quasi-drug of the present invention may be formulated into disinfectant cleansers, shower foams, liquid or solid ointments, wet wipes, coating agents, etc., but is not limited thereto, and the method of formulation, dosage, method of use, components, etc. of the quasi-drug may be appropriately selected from ordinary techniques known in the technical field.

[0102] The quasi-drug composition of the present invention can be used alone for the prevention or treatment of hypopigmentation-related diseases, and can be used in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers.

[0103] In the present invention, the cosmetic composition may be formulated as a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, paste, gel, suspension, emulsion, spray, cosmetic liquid, or capsule, but is not particularly limited thereto.

[0104] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers generally incorporated into cosmetic compositions for external use on the skin, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., may be appropriately incorporated, but is not limited thereto. The cosmetically acceptable carriers included in the cosmetic composition of the present invention may vary depending on the formulation.

[0105] When the cosmetic composition of the present invention is formulated into an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or a mixture thereof may be added as a carrier component.

[0106] When the cosmetic composition of the present invention is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0107] When the cosmetic composition of the present invention is formulated as a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be added as a carrier component, and more specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. may be added.

[0108] When the cosmetic composition of the present invention is formulated as a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be added as a carrier component.

[0109] When the cosmetic composition of the present invention is formulated into a capsule, it may be formulated in the form of an alginate capsule, an agar capsule, a gelatin capsule, a wax capsule, a double capsule, etc., but is not particularly limited thereto.

[0110] In addition, the cosmetic composition of the present invention may further include commonly used auxiliary agents, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, absorbents, and dyes, in addition to the aforementioned components.

[0111] In a specific embodiment of the present invention, a method for preventing, alleviating, or improving a hypopigmentation-related disease is provided, comprising the step of locally administering or applying a skin topical composition containing 2,3-dichloro-1,4-naphthoquinone (Diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient to the skin of a subject.

[0112] In a specific embodiment of the present invention, a health functional food composition for use in preventing, alleviating, or improving hypopigmentation-related diseases is provided, comprising 2,3-dichloro-1,4-naphthoquinone (Diclon), 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), or a mixture thereof as an active ingredient.

[0113] The terms "hypopigmentation-related disease," "prevention," and "alleviation" are as described above.

[0114] The term "improvement" as used herein may be used interchangeably with the aforementioned "alleviation" or "treatment," and refers to any phenomenon in which abnormal symptoms, disorders, or disease states caused by the excessive production or accumulation of pigment in cells or organs (e.g., the eye) are improved or beneficially altered by the health functional food composition of the present invention.

[0115] The “health functional food” of the present invention may further contain one or more known active ingredients that exhibit the effect of preventing, alleviating, or improving hypopigmentation-related diseases together with Diclon, Y15, or a mixture thereof.

[0116] In the present invention, "health functional food" refers to a functional food that helps prevent, alleviate, or improve diseases related to hypopigmentation, as well as aid in recovery to a normal state after experiencing related symptoms, and must be harmless to the human body when consumed over a long period.

[0117] In the present invention, when Diclon, Y15, or a mixture thereof is used as a food additive, the said ingredient may be added entirely alone, used together with other foods or food ingredients, or used appropriately according to conventional methods. The amount of the mixture of active ingredients may be appropriately determined according to the purpose of use, such as preventive treatment, health supportive treatment, or therapeutic treatment.

[0118] Generally, when manufacturing food or beverages, the diclon, Y15, or a mixture thereof of the present invention may be added in an amount of 15% by weight or less, 10% by weight or less, 5% by weight or less, 3% by weight or less, 1% by weight or less, etc., based on the total raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

[0119] The health functional food composition according to the present invention can be formulated in the form of pills, powders, liquids, capsules, or chewable films.

[0120] The health functional food composition of the present invention may include flavoring agents, sweeteners, preservatives, etc. as additional ingredients.

[0121] For example, natural sweeteners such as monosaccharides like glucose and fructose, disaccharides like maltose and sucrose, dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame can be used.

[0122] The health functional food composition of the present invention may include various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents, etc.

[0123] In a specific embodiment of the present invention, the present invention provides a composition for selectively inhibiting melanophage autophagy comprising a diclone, Y15, or a mixture thereof as an active ingredient.

[0124] In a specific embodiment of the present invention, a composition comprising a diclone, Y15, or a mixture thereof as an active ingredient is used to inhibit melanophages in melanocytes.

[0125] In a specific embodiment of the present invention, a composition comprising a diclone, Y15, or a mixture thereof as an active ingredient is used in the manufacture of a drug for treating pigmentation-related diseases.

[0126] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0127] According to the experimental examples and embodiments of the present invention, the utility of a novel signaling pathway regulating melanophages in melanocytes treated with 3,4,5-trimethoxycinnamate thymol ester (TCTE), which is known to effectively inhibit skin pigmentation by activating melanophages, was confirmed. In summary, ITCH (Itchy E3 ubiquitin ligase) promoted the ubiquitination of MLANA, a melanosomal membrane protein, during TCTE-induced melanophage, and ubiquitinated MLANA was found to be recognized by optineurin (OPTN), an autophagy adapter protein. Furthermore, through a phosphokinase antibody array, it was shown that TCTE phosphorylates ITCH to activate protein tyrosine kinase 2 (PTK2), which enhances the ubiquitination of MLANA. In addition, it was found that inhibition of PTK2 or ITCH inhibited ubiquitination of MLANA and MLANA-OPTN interactions in cells treated with TCTE. Taken together, the results of the study according to the present invention suggest that the role of the PTK2-ITCH-MLANA-OPTN cascade is important in coordinating the progression of melanophages.

[0128] The present invention will be explained in more detail below through experimental examples and embodiments. These experimental examples and embodiments are provided for the purpose of explaining the present invention more specifically, and the scope of the present invention is not limited to these experimental examples and embodiments.

[0129] Materials and Reagents

[0130] TCTE (3,4,5-trimethoxycinnamate thymol ester) was synthesized by Amorepacific researchers as previously described

[0026] . MG132 (474790), bafilomycin A1 (B1793), and diclon (CRM04144) were purchased from Sigma-Aldrich. Y15 (HY-12444) was purchased from MedChemExpress. Atg5(5'-ACCGGAAACUCAUGGAAUA-3'; SEQ ID NO. 1), Itch(5'-GACCUGAGAAGACGUUUGU-3'; SEQ ID NO. 2), Mlana(5'-GCUCUGCUUAUCGGCUGC-3'; SEQ ID NO. 3), Optn(5'-AGAGAGUGUUGGAAGCGAAGU-3'; SEQ ID NO. 4) and negative scrambled siRNA(5'-CCUACGCCACCAAUUUCGU-3'; SEQ ID NO. 5) were synthesized via Genolution.

[0131] Plasmid

[0132] To generate the pcDNA / TPC2-mRFP-EGFP plasmid, the PCR amplification products TPC2 and mRFP-EGFP were individually subcloned into pcDNA3.1 / Myc-His(-)A. The expression plasmids pCINeo-Myc-Itch, pOPTN-EGFP, pEGFP-TYR, mRFP-UBC, pEGFP-UBC, pRK5-HA-UBC-WT, pmRFP-LC3, and pGFP-Ptk2 were purchased from Addegene (11427, 27052, 32781, 11935, 11928, 17608, 21075, and 50515; deposited by Dr. Allan Weissman, Beatrice Yue, Ruth Halaban, Nico Dantuma, Ted Dawson, and Dr. Kenneth Yamada, respectively). pCMV3-MLANA-Flag(HG-12253-CF) was obtained from Sino Biological.

[0133] short interfering RNA (siRNA)

[0134] We self-constructed short interfering RNAs targeting melanosomal membrane proteins and melanophage adapters.

[0135]

[0136]

[0137] Experimental Example 1: B16F1 Cell Culture and Establishment of a Stable Cell Line

[0138] B16F1 cells were purchased from the American Type Culture Collection (CRL-6323). All cells were cultured at 37°C in a 5% CO2 incubator and maintained in DMEM containing 10% FBS and 1% penicillin / streptomycin (WELGENE, LM001-05, S001-07 and L502-02).

[0139] To generate stable cell lines, B16F1 melanoma cells were transfected with pcDNA / TPC2-mRFP-EGFP (B16F1 / TPC2-mRFP-EGFP) using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer's protocol. Stable transfected cells were selected in screening medium containing 1.25 mg / ml of G418 (Invitrogen, 11811023) for 10 days. After inoculating individual cells, stable clones were selected using a fluorescence microscope (Olympus, IX70, Tokyo, Japan).

[0140] Experimental Example 2: Melan-a Cell Culture and Stable Maintenance

[0141] Melan-a melanocytes were obtained from Dr. Dorothy C. Bennett (ST. George's Hospital Medical School, London, BK) and maintained in RPMI 1640 medium (WELGENE, LM011-01) supplemented with 10% FBS, 1% P / S, and 200 nM phorbol-12-myristate-13-acetate (Sigma, P8139) in a 5% CO2 humidified incubator at 37°C.

[0142] Experimental Example 3: Western Blotting

[0143] All lysates were prepared using 2Х Laemmli sample buffer (Bio-Rad, 1610737). Total protein was quantified using Bradford solution (Bio-Rad, 5000001) according to the manufacturer's instructions. The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, 1620177). After blocking the membrane with 4% skim milk (MBcell, MB-4667) in TBST (25 mM Tris-base, 140 mM NaCl [GenDEPOT, T9200 and G0610] and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the indicated primary antibodies: anti-LC3 (NB100-2220; 1:3000) and anti-ATG5 (NB110-53818; 1:3000) antibodies purchased from NOVUS biologicals; anti-Itch (611198; 1:1000) purchased from BD Biosciences; Anti-Myc (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; anti-OPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5-15257, 1:1000), purchased from Invitrogen; The membrane was incubated with anti-β-actin (660009-1-Ig; 1:10000) purchased from Proteintech. The membrane was incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).The signal was detected using Luminograph II (ATTO, Tokyo, Japan).

[0144] Experimental Example 4: Immunoprecipitation

[0145] For immunoprecipitation analysis, cells were homogenized overnight at 4°C in RIPA buffer containing a protease inhibitor (GenDEPOT) (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and 2 mM EDTA; iNtRON Biotechnology, IBS-BR002). For exogenous IP analysis, the supernatant was immunoprecipitated with the following antibodies: anti-Flag-agarose antibody (Abcam, ab1240), anti-Myc-agarose (Santa Cruz Biotechnology, SC-40 AC), and monoclonal anti-phosphorusherin-agarose antibody (Sigma-Aldrich, A8076). After incubation overnight, the samples were washed twice with RIPA buffer at 4°C, and then 2x Laemmli sample buffer (Bio-Rad) was added. All samples were analyzed by Western blotting as described in the Western blotting section.

[0146] Experimental Example 5: In Vitro Ubiquitination Analysis

[0147] In vitro ubiquitination analysis was performed using the ubiquitination analysis kit (Abcam, ab139467) according to the manufacturer's instructions. HEK293T cells were transfected with Myc or Myc-Itch. After 24 hours, the cells were homogenized and precipitated with anti-Myc-agarose (Santa Cruz Biotechnology) using the same method as for the immunoprecipitation section. Purified Myc or Myc-Itch was incubated with a reaction solution (0.1 μM E1 [Abcam], 6.6 μg UbcH7 / UBE2L3 [R&D System, E2-640-100], 1.25 μg MLANA protein [Proteintech, Ag13387], 2.5 μM Ub [Abcam], 0.02 U / μl inorganic pyrophosphatase solution [Thermofisher, EF0221], 1 mM dithiothreitol [Thermofisher, R0861], 0.1 Mg-ATP [Abcam], 5 mM EDTA [GenDEPOT, P3100-001]) at 37°C for 2 hours. After incubation, the reaction was terminated with 2X non-reducing gel loading buffer (Abcam), and ubiquitination was analyzed by Western blotting.

[0148] Experimental Example 6: Confocal Microscope Observation

[0149] B16F1 cells were washed twice with phosphate-buffered saline (WELGENE) at 37°C and then fixed with 4% paraformaldehyde (Biosesang) at room temperature for 15 minutes. To increase permeability, cells were incubated with 0.1% Triton X-100 for 5 minutes and blocked with 2% BSA in PBS for 1 hour at room temperature. After blocking, cells were incubated overnight at 4°C with a primary antibody against anti-Flag (Sigma-Aldrich, F1804). Then, cells were washed three times with PBS and incubated with Alexa Fluor 555 Goat anti-mouse IgG antibody (Thermo Fisher, A21422) in a dark room at room temperature for 1 hour. Cells were washed three times with PBS, and glass coverslips containing cells were placed on glass slides using anti-fade fluorescent mounting medium (Abcam, 104135). Fluorescence images were captured using a confocal laser scanning microscope (Carl Zeiss, LSM800, NY, USA) and processed with ZEISS Zen software.

[0150] Experimental Example 7: Quantification of Melanophage

[0151] To quantify cells containing melanophages, B16F1 / TPC2-mRFP-EGFP cells were grown on cover glass and transfected with siRNAs for Mlana, Optn, and Itch. Then, the cells were treated with α-MSH (1 μM, 72 hours) or with TCTE (10 μg / ml, 24 hours). Subsequently, the cells were washed with PBS (WELGENE) and fixed with 4% paraformaldehyde (Biosesang) for 20 minutes. Fluorescence images were then obtained using a confocal laser scanning microscope (Carl Zeiss). The number of melanosomes containing only mRFP signals was counted using ImageJ (NIH) on EGFP and mRFP-combined images.

[0152] Experimental Example 8: Proteome Profiler Human Phosphor-Kinase Array Analysis

[0153] To perform PPHPK (Proteome Profiler Human Phosphor-Kinase) array (R&D Systems, ARY003C) analysis, B16F1 cells stimulated with α-MSH were treated with TCTE (10 μg / ml) for 24 hours. Cells were washed with cold PBS and lysed in cell lysis buffer containing a protease and phosphatase inhibitor cocktail at 4°C for 30 minutes. Then, the phosphorylated antibody array was incubated overnight with the supernatant at 4°C. The array was washed and incubated with the detection antibody cocktail and HRP-conjugated anti-rabbit IgG, respectively, at room temperature for 3 hours. Chemiluminescent signals were detected with the detection buffer using Luminograph II (ATTO).

[0154] Experimental Example 9: Cell-based ubiquitination compound library screening

[0155] To perform cell-based ubiquitination compound library screening, B16F1 / TPC2-mRFP-EGFP cells stimulated with α-MSH were seeded in 96-well plates. After 48 hours of culture, the cells were treated with the TargetMol ubiquitination compound library (TargetMol, L8600), which contains 78 small molecules targeting various components of the ubiquitination pathway including proteasomes, E1 / E2 / E3 enzymes, DUB, and p97, as identified in the 'List of Chemicals in the Ubiquitination Compound Library' shown in Table 2, at various concentrations (5-100 μM). Co-treatment with α-MSH (1 μM) and TCTE (10 μg / ml) was performed in each well. After 24 hours, cells were fixed with 4% paraformaldehyde (Biosesang, P2031) and monitored using an Operetta CLS™ High-content assay system (Perkin Elmer, MA, USA). The proportion of cells containing melanophages was evaluated and classified into relative grades ranging from 1 to 5. Grade 1 indicates that less than 10% of cells contain melanophages (e.g., cells treated with TCTE and bafilomycin A1), 10 to 30% is Grade 2, 30 to 60% is Grade 3, 60 to 80% is Grade 4, and more than 80% is Grade 5 (e.g., cells treated with TCTE only).

[0156]

[0157] Experimental Example 10: Melanin Content Analysis

[0158] To measure melanin content, B16F1 cells were transfected with scrambled siRNA (Sc), Itch-targeted siRNA (siItch), Mlana-targeted siRNA (siMlana), or Optn-targeted siRNA (siOptn). After 1 day, cells were treated with α-MSH (1 μM) for 48 hours and additionally exposed to TCTE (10 μg / ml) for 24 hours. Then, cells were harvested by trypsin treatment and lysed in solubilization buffer at 100°C for 30 minutes. Relative melanin content was determined by measuring at 415 nm using an ELISA plate reader (Biotek, VT, USA).

[0159] Statistical analysis

[0160] Data were collected through at least three independent experiments and expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism 9 software. Results were statistically analyzed using the t-test. * A p < 0.001 value was considered significant to the data.

[0161] result

[0162] Example 1: ITCH mediates TCTE-induced melanophage in B16F1 cells

[0163] The inventors recently revealed that TCTE is a potent inducer of melanophages in hyperpigmented cells

[0029] . However, the molecular mechanism underlying melanophages is little known. Through the present invention, the mechanism of melanophages was investigated in more detail. Since ubiquitination of target organelles acts as a tagging process indicating organelle-selective degradation, we first investigated whether melanosomes are ubiquitinated during melanophage.

[0164] As can be seen in Fig. 1a, TCTE treatment significantly increased the co-localization coefficient between tyrosinase and ubiquitin in B16F1 cells stimulated with α-MSH. In addition, TCTE enhanced the co-localization of tyrosinase and LC3, which are autophagosome markers (Fig. 4).

[0165] To investigate TCTE-mediated melanophages in greater depth, the inventors used a monitoring system (TPC2-mRFP-EGFP) handling two-pore segment channel 2 (TPC2) proteins linked with tandem fluorescent tags [29,31,32]. In B16F1 / TPC2-mRFP-EGFP cells treated with TCTE, the number of red-only spots significantly increased, indicating a decrease in the green signal (mRFP[+] / EGFP[-]) in autolysosomes. However, this mRFP-positive signal was significantly inhibited by Atg5 knockdown, implying that TCTE induces melanosome degradation through autophagy activation in B16F1 cells stimulated with α-MSH (Fig. 1b). In addition, LC3-II protein levels were elevated in cells treated with TCTE and were blocked by the depletion of Atg5 (Fig. 1c).

[0166] To further investigate the molecular mechanisms of melanophages, the inventors screened a library of ubiquitinated compounds with TCTE and identified Diclon (2,3-dichloro-1,4-naphthoquinone) as the most potent novel melanophage regulator (Fig. 5). To verify these findings, B16F1 / TPC2-mRFP-EGFP cells were treated with Diclon in combination with TCTE. As shown in Fig. 1d, Diclon treatment significantly blocked the TCTE-enhanced RFP-positive signal in B16F1 cells stimulated with α-MSH. It was previously reported that 1,4-naphthoquinone, a derivative of Diclon, inhibits the HECT domain E3 ligase, ITCH

[0033] . Accordingly, the inventors further investigated the role of ITCH in melanophages. The increase in mRFP+ / EGFP- spot signaling induced by TCTE was reduced by the depletion of ITCH expression in B16F1 cells stimulated with α-MSH (Fig. 1e, Fig. 8A). Additionally, the depigmentation effect of TCTE was attenuated by the downregulation of Itch (Fig. 9A). These results collectively indicate that inhibition of ITCH reduces melanophages in cells treated with TCTE.

[0167] Example 2: MLANA ubiquitinated by ITCH interacts with OPTN to induce melanophages in cells treated with TCTE.

[0168] To better understand the role of ITCH in melanophages, the inventors identified potential ubiquitination targets among melanosomal membrane proteins in TCTE-treated cells. The inventors constructed a small siRNA library using various melanosomal membrane proteins and identified MLANA as a novel regulator of melanophages through library screening (Fig. 6). To verify these results, the inventors used B16F1 / TPC2-mRFP-EGFP cells, depleted MLANA via RNA interference, and treated them with TCTE. As shown in Fig. 2a, the downregulation of MLANA efficiently suppressed mRFP+ / EGFP- spot signaling in cells treated with TCTE after α-MSH stimulation (Fig. 2a, Fig. 8B). Nevertheless, the depigmentation activity of TCTE was not significantly affected by the downregulation of MLANA (Fig. 9B).

[0169] Next, the inventors investigated whether MLANA is a substrate of ITCH. Importantly, immunoprecipitation analysis revealed that ITCH overexpression was associated with MLANA in B16F1 cells stimulated with α-MSH (Fig. 2b). The co-localization coefficient between MLANA and ubiquitin was significantly enhanced in B16F1 cells treated with TCTE (Fig. 2c). Furthermore, ubiquitination of MLANA increased significantly in response to TCTE treatment compared to untreated control cells (Fig. 2d). Additionally, ubiquitination analysis revealed that ubiquitination of purified MLANA protein increased upon ITCH supplementation (Fig. 2e). Moreover, Itch depletion reduced ubiquitination of MLANA in B16F1 cells treated with TCTE (Fig. 2f), suggesting that MLANA is a target of ITCH.

[0170] Next, the inventors investigated the autophagy adapters involved in recognizing ubiquitinated MLANA during melanophage. Downregulation of NBR1 or SQSTM1 did not result in significant changes in melanophage after TCTE treatment in B16F1 cells stimulated with α-MSH (Fig. 7). However, depletion of OPTN almost completely reversed the increase in red spot signals, suggesting that OPTN is primarily involved in the recognition of ubiquitinated melanosomes (Fig. 2g, Fig. 8C). Additionally, the whitening effect of TCTE was reduced by the knockdown of OPTN (Fig. 9C). Further immunoprecipitation analysis revealed that MLANA interacts with OPTN in B16F1 cells treated with TCTE (Fig. 2h). Collectively, the results according to the present invention indicate that MLANA is ubiquitinated by ITCH and promotes melanophage in B16F1 cells treated with TCTE in cooperation with OPTN.

[0171] Example 3: When PTK2 is activated in response to TCTE, ITCH is phosphorylated to promote melanophage in B16F1 cells.

[0172] The inventors further investigated the upstream regulatory mechanisms affecting ITCH activity. Analysis using a phosphokinase antibody array revealed that PTK2 was activated after treatment with TCTE (Fig. 3a). This activation was confirmed by Western blotting analysis, which showed phosphorylation of PTK2 at tyrosine 397 in response to TCTE treatment (Fig. 3b). The inventors investigated the effects of PTK2 phosphorylation on melanophages in B16F1 cells treated with TCTE. As shown in Fig. 3c, treatment with Y15, a potent and specific chemoinhibitor of PTK2, significantly reduced mRFP+ / EGFP- spot signals in TCTE-treated cells (Fig. 3c). Furthermore, the inventors discovered that PTK2 corresponds to ITCH in response to TCTE stimulation (Fig. 3d), implying that activation is important in TCTE-mediated melanophages.

[0173] Next, the inventors investigated the upstream regulatory mechanisms controlling ITCH activity and confirmed that ITCH is phosphorylated upon TCTE treatment, and that this process was significantly attenuated by the simultaneous application of the PTK2 inhibitor Y15 (Fig. 3e). Considering the important role of PTK2 in mediating ITCH phosphorylation after TCTE treatment, the interaction with MLANA, and the subsequent ubiquitination and recruitment of the autophagy adapter OPTN, the inventors investigated the roles of PTK2 and ITCH in the ubiquitination of MLANA and OPTN recruitment. Notably, inhibiting PTK2 or ITCH using Y15 and Diclon reduced the increase in MLANA ubiquitination in TCTE-treated cells (Fig. 3f). From Figs. 3e and 3f, it can be inferred that Y15 and Diclon are involved in the regulation of melanophages by blocking the phosphorylation of ITCH or inhibiting ITCH activity.

[0174] In addition, consistently, the interaction between MLANA and OPTN, enhanced by TCTE treatment in α-MSH-stimulated B16F1 cells, was inhibited by Y15 and diclon treatment (Fig. 3g). Collectively, the inventors' findings highlight the important role of the PTK2-ITCH-MLANA-OPTN signaling cascade in the modulation of melanophages (Fig. 3h).

[0175] Discussion

[0176] Melanophages regulate melanin levels by breaking down melanosomes, which are important for skin pigmentation and UV protection. An imbalance between the production and breakdown of melanosomes can lead to pigmentation disorders, which highlights the importance of melanosomes in skin health [23,30,34]. Previously, the inventors discovered that TCTE promotes depigmentation by increasing the autophagic degradation of intracellular melanosomes

[0029] . However, the precise regulatory mechanisms underlying melanophages are largely unknown. The present invention provides comprehensive insights into the molecular complexity of melanophages. Among selective autophagic processes, mitophagy is the most well-defined. Mitophagy involves the detection of PINK1 kinase and the E3 ligase Parkin ubiquitination of mitochondrial outer membrane proteins to tag damaged mitochondria, and the ubiquitinated proteins are recognized by autophagosome adapter proteins to recruit autophagosomes

[0035] . According to this mitophagy regulation scenario (PINK1-Parkin-VDAC1-SQSTM1), the inventors' findings suggest that the PTK2-ITCH-MLANA-OPTN cascade plays a crucial role in melanophage regulation. This novel cascade not only provides a new pathway for understanding melanosome degradation but also offers potential therapeutic targets for conditions associated with changes in skin pigmentation.

[0177] Through the research results according to the present invention, the inventors confirmed that the initiation of the melanophage pathway is associated with the activation of PTK2, as shown in Fig. 3. Although further research is needed to confirm the specific role of PTK2 in melanophages, the interaction between peroxisome proliferator-activated receptor gamma (PPARγ) and PTK2 is presumed to be significant. Recent studies suggest that TCTE can act as a PPARγ agonist, which is supported by the reduction in depigmentation observed when using PPARγ antagonists

[0036] . Furthermore, the decrease in PTK2 phosphorylation in the presence of PPARγ antagonists suggests the possibility of PPARγ-mediated activation of PTK2 [36, 37]. Therefore, further investigation is required to elucidate the underlying mechanism of this interaction.

[0178] Although we cannot identify the target residue of ITCH mediated by PTK2, it is clear that the phosphorylation of ITCH by PTK2 is a critical event that promotes the subsequent ubiquitination of MLANA. This ubiquitination is necessary for melanosomes to become targets for autophagic degradation. Following ITCH-mediated ubiquitination, the interaction between MLANA and OPTN represents a critical juncture for recruiting melanosomes to the autophagic machinery (Fig. 3). This process is further supported by the observation that melanophages are inhibited by ITCH deficiency, which highlights the importance of ITCH's role in this pathway. Furthermore, the inventors' findings provide further validation of the melanophage cascade and extend beyond cancer cell lines to include non-cancer cell lines such as Melan-A cells (Fig. 10). Thus, the inventors' findings broaden the understanding of ITCH regulation and highlight the complexity of signaling pathways associated with melanophages.

[0179] The convergence of autophagy and melanogenesis is important not only for understanding the physiological processes governing skin pigmentation but also for pathological conditions [23,30]. For example, hyperpigmentation disorders can be better understood and managed through targeted interventions that modulate this pathway. The therapeutic potential of melanophage manipulation is particularly interesting. It may be possible to develop new strategies for treating pigmentation disorders by targeting specific components of the PTK2-ITCH-MLANA-OPTN cascade. For example, modulation of ITCH activity or disruption of the interaction between ITCH and MLANA, as presented herein, could be a new approach to regulating melanin levels in hyperpigmented skin.

[0180] According to the inventors' research results, the reduction in melanin in cells treated with TCTE was reversed by the downregulation of ITCH or OPTN, whereas this was not the case with MLANA (Fig. 9). This is presumed to be due to the role of MLANA in regulating melanosomal matrix proteins and premlanosomal proteins for melanosome maturation. Further research is required to understand the precise mechanism of MLANA in melanosome biogenesis. Furthermore, PTK2 inhibitors may provide another pathway for regulating melanophages and, by extension, skin pigmentation. However, the specific effect of TCTE on melanophages revealed by the research results according to the present invention may differ from that of other compounds or other physiological conditions. Therefore, future research should aim to verify these results in vivo and explore potential side effects and efficacy targeting this pathway in a clinical setting.

[0181] In summary, the inventors concluded that the PTK2-ITCH-MLANA-OPTN cascade is an important melanophage regulatory cascade that can broaden the understanding of melanosome degradation mechanisms and present new strategic approaches for skin pigmentation disorders. Based on the newly identified novel melanophage regulatory signaling cascade, the inventors established a screening method for substances with melanophage inhibitory activity. This not only improves the understanding of melanophage regulatory mechanisms but also allows for the efficient identification of the usefulness and potential utility of any substance as a melanophage inhibitor in a short period of time. Therefore, the present invention is expected to have high utility value as a platform technology for melanophage regulation.

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[0223] The present invention can easily determine the efficacy and utility of substances having melanophage inhibitory activity based on the PTK2-ITCH-MLANA-OPTN cascade, a newly identified melanophage regulatory signaling pathway. The present invention can provide substances with excellent preventive, alleviative, improvement, or therapeutic effects for diseases related to hypopigmentation. By providing an in-depth understanding of melanophage-related abnormalities or disorders, the present invention can present a new approach to developing innovative therapies and cosmetic strategies to address the problem of hypopigmentation.

Claims

1. A screening method for substances having melanophage inhibitory activity, a. A step of preparing B16F1 melanoma cells into which an expression cassette comprising mRFP (monomeric Red Fluorescent Protein) and EGFP (Enhanced Green Fluorescent Protein) operably linked to TPC2 (Two pore segment channel 2) has been introduced; b. A step of inducing melanophages in prepared B16F1 melanoma cells by treating them with a melanophage-inducing substance without or with a test substance, and stimulating the cells by treating them with alpha-melanocyte-stimulating hormone (α-MSH); c. Fixing the cells, then capturing a fluorescence image of the cells to generate an image that merges the red fluorescence of mRFP and the green fluorescence of EGFP, and measuring the number of puntas exhibiting red fluorescence from the generated image; d. A step of determining a test substance that reduces the number of spots exhibiting red fluorescence compared to the case where a melanophage-inducing substance is treated without the test substance as an effective substance having melanophage-inhibiting activity; comprising a method.

2. In Claim 1, A screening method for a substance having melanophage inhibitory activity, characterized in that the melanophage-inducing substance is a substance that exhibits whitening activity based on a melanophage pathway including 5-trimethoxycinnamate thymol ester (TCTE).

3. In Claim 1, A screening method for substances having melanophage inhibitory activity, characterized in that, in step b, melanophages are induced through a PTK2-ITCH-MLANA-OPTN signaling cascade consisting of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment.

4. In Claim 1, A screening method for substances having melanophage inhibitory activity, characterized in that, in step c, a fluorescence image of the cell is obtained using a confocal fluorescence microscope.

5. In Claim 1, A screening method for a substance having melanophage inhibitory activity, characterized in that, in step d, the number of spots exhibiting red fluorescence when treated with a melanophage-inducing substance together with a test substance is reduced by at least 30% compared to the number of spots exhibiting red fluorescence when treated with a melanophage-inducing substance without a test substance, is determined to be a substance having effective melanophage inhibitory activity.

6. In Claim 4, A screening method for substances having melanophage inhibitory activity, characterized in that a substance determined to be an effective substance inhibits melanosome degradation.

7. In Claim 3, A screening method for a substance having melanophage inhibitory activity, comprising the step of further determining whether a substance determined to be an effective substance attenuates or blocks at least one signaling pathway of the PTK2-ITCH-MLANA-OPTN signaling cascade, which consists of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment.

8. In Claim 7, A screening method for a substance having melanophage inhibitory activity, characterized in that a substance determined to be an effective substance inhibits ITCH (Itchy E3 ubiqutin ligase) activity, inhibits the phosphorylation of ITCH, or weakens or inhibits the interaction between ITCH and MLANA (Melan-A).

9. In Claim 8, A screening method for a substance having melanophage inhibitory activity, characterized in that the substance determined to be an effective substance is 2,3-dichloro-1,4-naphthoquinone or 1,2,4,5-benzenetetraamine tetrahydrochloride.

10. In claim 7 or 9, A screening method for a substance having melanophage inhibitory activity, characterized in that a test substance determined to be an effective substance can verify whether the melanophage-inducing substance treated in step b) exhibits whitening activity through the PTK2-ITCH-MLANA-OPTN signaling cascade signaling pathway, which consists of i) PTK2 (Protein Tyrosine Kinase 2) activation, ii) ITCH (Itchy E3 ubiqutin ligase) phosphorylation, iii) MLANA (Melan-A) ubiquitination, and iv) OPTN (Optineurin)-mediated autophagy adapter protein recruitment. A pharmaceutical composition for use in preventing, alleviating, or treating hypopigmentation-related diseases, comprising as an active ingredient 11.2,3-dichloro-1,4-naphthoquinone, 1,2,4,5-benzenetetraamine tetrahydrochloride, or a mixture thereof.

12. In Claim 11, A pharmaceutical composition characterized in that the hypopigmentation-related disease is any one selected from the group consisting of vitiligo, albinism, partial albinism, depigmented nevus, chemical-induced white skin disease, idiopathic guttate hypomelanosis, punctate white skin disease, Freddy-Willi syndrome, Waddenbrook syndrome, Vogt-Koyanagi-Harada syndrome, fungal infection (tinea versicolor), pityriasis whiteis (ringworm), and Ito hypomelanosis. A topical skin composition for use in preventing, alleviating, or improving hypopigmentation-related diseases, comprising as an active ingredient 13.2,3-dichloro-1,4-naphthoquinone, 1,2,4,5-benzenetetraamine tetrahydrochloride, or a mixture thereof.

14. In Claim 13, A topical skin composition characterized by the hypopigmentation-related disease being any one selected from the group consisting of vitiligo, albinism, partial albinism, depigmented nevus, chemical-induced white skin disease, idiopathic guttate hypomelanosis, punctate white skin disease, Freddy-Willi syndrome, Waddenbrook syndrome, Vogt-Koyanagi-Harada syndrome, fungal infection (tinea versicolor), pityriasis whiteis (ringworm), and Ito hypomelanosis.

15. In Claim 14, A topical skin composition characterized by being formulated as a quasi-drug or cosmetic. A health functional food composition for use in preventing, alleviating, or improving pigmentation-related diseases, comprising 16.2,3-dichloro-1,4-naphthoquinone, 1,2,4,5-benzenetetraamine tetrahydrochloride, or a mixture thereof as an active ingredient.

17. In Claim 16, A health functional food composition characterized in that the hypopigmentation-related disease is any one selected from the group consisting of vitiligo, albinism, partial albinism, depigmented nevus, chemical-induced white skin disease, idiopathic guttate hypomelanosis, punctate white skin disease, Freddy-Willi syndrome, Waddenbrook syndrome, Vogt-Koyanagi-Harada syndrome, fungal infection (tinea versicolor), pityriasis whiteis (ringworm), and Ito hypomelanosis.

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