Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A.

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.

Anthocyanins in perilla plants and dried leaves.

High-quality perilla leaves are purple on upper and lower surfaces and have a good aroma. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not that of anthocyanins. Several reports have described the chemical species of anthocyanins in red perilla, but a complete analysis of anthocyanins in perilla has not been reported. In this study, the anthocyanins in the leaves of cultivated and wild species of perilla and those in commercially available perilla herbs were studied. Red perilla and most P. citriodora strains accumulate cyanidin derivatives that differ in the acyl group on the glucose moiety at the 3-O- and 5-O-positions of the anthocyanins. Several strains of P. citriodora contain cyanidin derivatives that are different from those in red perilla and most P. citriodora species. Green perilla and wild species other than P. citriodora do not contain foliar anthocyanins. The anthocyanins in commercially available perilla herbs and natural dyes made from red perilla were in agreement with those in fresh red perilla leaves and most P. citriodora samples. The amounts and types of anthocyanins were not associated with place of cultivation, although some changes occurred due to degradation during storage. These results provide clues regarding the biosynthesis of anthocyanins in perilla and the evolution of red perilla. The characteristics and stability of anthocyanins are discussed.