Identification of novel neuroprotective N,N-dimethylaniline derivatives that prevent oxytosis/ferroptosis and localize to late endosomes and lysosomes.

Oxidative stress has been implicated in the aging process and the progression of many neurodegenerative disorders. We previously reported that a novel oxindole compound, GIF-0726-r, effectively prevents endogenous oxidative stress, such as oxytosis/ferroptosis, an iron-dependent form of non-apoptotic cell death, in mouse hippocampal cells. In this study, using two hundred compounds that were developed based on the structure-activity relationship of GIF-0726-r, we screened for the most potent compounds that prevent glutamate- and erastin-induced oxytosis and ferroptosis. Using submicromolar concentrations, we identified nine neuroprotective compounds that have N,N-dimethylaniline as a common structure but no longer contain an oxindole ring. The most potent derivatives, GIF-2114 and GIF-2197-r (the racemate of GIF-2115 and GIF-2196), did not affect glutathione levels, had no antioxidant activity in vitro, or ability to activate the Nrf2 pathway, but prevented oxytosis/ferroptosis via reducing reactive oxygen production and decreasing ferrous ions. Furthermore, we developed fluorescent probes of GIF-2114 and GIF-2197-r to image their distribution in live cells and found that they preferentially accumulated in late endosomes/lysosomes, which play a central role in iron metabolism. These results suggest that GIF-2114 and GIF-2197-r protect hippocampal cells from oxytosis/ferroptosis by targeting late endosomes and lysosomes, as well as decreasing ferrous ions.

Zebrafish as a new model for rhododendrol-induced leukoderma.

Idiopathic leukoderma is a skin disorder characterized by patchy loss of skin pigmentation due to melanocyte dysfunction or deficiency. Rhododendrol (RD) was approved as a cosmetic ingredient in Japan in 2008. However, it was shown to induce leukoderma in approximately 20,000 customers. The prediction of cytotoxicity, especially to melanocytes in vivo, is required to avoid such adverse effects. Since the use of higher vertebrates is prohibited for medicinal and toxicological assays, we used zebrafish, whose melanocytes were regulated by mechanisms similar to mammals. Zebrafish larvae were treated with RD in breeding water for 3 days, which caused body lightening accompanied by a decrease in the number of melanophores. Interestingly, black particles were found at the bottom of culture dishes, suggesting that the melanophores peeled off from the body. In addition, RT-PCR analysis suggested that the mRNA levels of melanophore-specific genes were significantly low. An increase in the production of reactive oxygen species was found in larvae treated with RD. The treatments of the fish with other phenol compounds, which have been reported to cause leukoderma, also induced depigmentation and melanophore loss. These results suggest that zebrafish larvae could be used for the evaluation of leukoderma caused by chemicals, including RD.

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.

The High Content of Ent-11α-hydroxy-15-oxo-kaur- 16-en-19-oic Acid in Adenostemma lavenia (L.) O. Kuntze Leaf Extract: With Preliminary in Vivo Assays.

BACKGROUND: Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA) is a multifunctional biochemical found in some ferns, Pteris semipinnata, and its congeneric species. Although a number of therapeutic applications of 11αOH-KA have been proposed (e.g., anti-cancer, anti-inflammation, and skin whitening), the content of 11αOH-KA in these ferns is not high. Adenostemma lavenia (L.) O. Kuntze, an Asteraceae, has also been reported to contain 11αOH-KA. The decoction (hot water extract) of whole plants of A. lavenia is used as a folk remedy for inflammatory disorders, such as hepatitis and pneumonia, suggesting that 11αOH-KA may be the ingredient responsible for the medicinal properties of this plant. METHODS: The anti-melanogenic activities of the water extracts of A. lavenia leaves and Pteris dispar Kunze (a cognate of P. semipinnata) leaves were compared in mouse B16F10 melanoma cells. The amount of 11αOH-KA was measured by using liquid chromatography spectrometry. C57BL/6J mice were treated with the water extract of A. lavenia leaf, and the blood concentration of 11αOH-KA was measured. The in vivo efficacy of the water extract of A. lavenia leaf was evaluated according to tis anti-melanogenic activity by monitoring hair color. RESULTS: Although both the extracts (A. lavenia and P. dispar Kunze) showed high anti-melanogenic activities, only A. lavenia contained a high amount of 11αOH-KA, approximately 2.5% of the dry leaf weight. 11αOH-KA can be purified from A. lavenia leaves in two steps: water extraction followed by chloroform distribution. The treatment of mice with the water extract of A. lavenia leaf suppresses pigmentation in their hairs. CONCLUSIONS: Despite the small number of mice examined, the present preliminary result of the suppressed hair pigmentation suggests that the water extract of A. lavenia leaf and the ingredient that is possibly responsible for this-11αOH-KA-are new materials for oral cosmetics. The results may also be helpful in the future development of functional foods and methods to treat patients suffering from hyperpigmentation disorders, such as melasma.