Thioxothiazolidin derivative, 4-OST, inhibits melanogenesis by enhancing the specific recruitment of tyrosinase-containing vesicles to lysosome.

Tyrosinase catalyzes the rate-limiting step in melanin synthesis. Melanin is synthesized from l-tyrosin in the melanosomes, where tyrosinase and other melanogenic factors are recruited via the vesicle transport system. Genetic and biochemical approaches have revealed a correlation between impairments in the vesicle transport system and albinism. However, the specificity of the individual transport systems for the corresponding melanogenic factors has not been well elucidated yet. Here, we report that the thioxothiazolidin derivative, 4-OST (4-[(5E)-5-[(4-fluorophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-4-azatricyclo [5.2.1.02 ,6]dec-8-ene-3,5-dione: CAS RN. 477766-87-3) strongly inhibited melanogenesis in mouse melanoma B16F10 cells. 4-OST reduces tyrosinase protein levels without affecting its messenger RNA levels or enzymatic activity. Although a reduction in tyrosinase protein level was observed in the presence of a protein synthesis inhibitor, the reduction may be coupled with protein synthesis. Similarly, GIF-2202 (a derivative of 4-OST) lowers tyrosinase protein levels without affecting the levels of another melanogenic enzyme, tyrosinase-related protein 1 (TYRP1) level. The reduction in tyrosinase protein level is associated with an increase in the levels of the lysosomal proteinase cathepsin S. Chloroquine, a lysosome inhibitor, restored the tyrosinase protein level downregulated by GIF-2202, although no effects of other inhibitors (against proteasome, autophagy, or exocytosis) were observed. In addition, GIF-2202 segregated the immunofluorescence signals of tyrosinase from those of TYRP1. Chloroquine treatment resulted in co-localization of tyrosinase and cathepsin S signals near the perinuclear region, suggesting that 4-OST and GIF-2202 may alter the destination of the tyrosinase vesicle from the melanosome to the lysosome. 4-OST and GIF-2202 can be new tools for studying the tyrosinase-specific vesicle transport system.

Azepine derivative T4FAT, a new copper chelator, inhibits tyrosinase.

Melanin plays an important role in the protection of the skin from ultraviolet irradiation. However, excessive melanin deposition leads to hyperpigmentation and freckles, which are recognized as skin problems, and signs of aging. Tyrosinase, a copper-containing protein, is the rate-limiting enzyme in melanin biosynthesis and first catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the further oxidization to dopaquinone. To assist the proper regulation of melanin production, we screened compounds and found that 5,6,7,8-tetrahydro-4H-furo[3,2-c]azepine-4-thione (T4FAT), a thioamide derivative, inhibited melanogenesis in B16F10 mouse melanoma cells. T4FAT was not toxic to cells and was stable in water; in addition, it inhibited the activity of tyrosinase derived from mushroom and B16F10 cells in a non-competitive manner. T4FAT downregulated tyrosinase protein expression in B16F10 cells without affecting mRNA expression. As copper binding to the tyrosinase protein is required for both enzymatic activity, correct folding, and maturation, we examined the metal-chelating activities of T4FAT. Equimolar amount of T4FAT resulted in almost complete chelation of copper ions. The thioamide group of T4FAT is essential for copper chelation and tyrosinase inhibition, which subsequently resulted in melanogenesis inhibition in B16F10 cells. Although T4FAT has similar in vitro properties to kojic acid, which is also a copper chelator and approved as a component of cosmetic formulations, T4FAT inhibited melanogenesis in B16F10 cells 30 times more efficiently than kojic acid. These results suggested that T4FAT, a novel copper chelator, may be helpful for the development of new cosmetics for skin whitening.