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Anithiactin D (<b>1</b>), a 2-phenylthiazole class of natural products, was isolated from marine mudflat-derived actinomycetes <i>Streptomyces</i> sp. 10A085. The chemical structure of <b>1</b> was elucidated based on the interpretation of NMR and MS data. The absolute configuration of <b>1</b> was determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectral data. Anithiactin D (<b>1</b>) significantly decreased cancer cell migration and invasion activities at a concentration of 5 μM via downregulation of the epithelial-to-mesenchymal transition (EMT) markers in A549, AGS, and Caco-2 cell lines. Moreover, <b>1</b> inhibited the activity of Rho GTPases, including Rac1 and RhoA in the A549 cell line, suppressed RhoA in AGS and Caco-2 cell lines, and decreased the mRNA expression levels of some matrix metalloproteinases (MMPs) in AGS and Caco-2 cell lines. Thus <b>1</b>, which is a new entity of the 2-phenylthiazole class of natural products with a unique aniline-indole fused moiety, is a potent inhibitor of the motility of cancer cells.