Alkamides from Echinacea disrupt the fungal cell wall-membrane complex.

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.