Secondary Assays for Testing the Mode of Action of Natural Products with Bioactivity Against Fungi

Identification of novel antifungals is needed, and plant-derived compounds offer important leads in this respect. This article initiates a compendium of secondary assays for testing hypotheses on mode of action of plant-derived compounds. Three assays were developed to evaluate compounds that are hypothesized to effect cell wall function, transcription, and oxidative stress. A hypothesized perturbation on cell wall integrity by chimaphilin and Echinacea root extracts was supported using sonication and heat shock assays. Heat shock treatments reduced the number of viable cells recovered when yeast was grown in the presence of subinhibitory concentrations of chimaphilin. A reduction in cell numbers was also observed with both sonication-based and heat shock-based assays when yeast was grown in the presence of Echinacea purpurea (L.) Moench (Asteraceae) extracts. The hypothesis that chimaphilin impedes mRNA transcription processes was also supported by a β-galactosidase reporter gene assay. The antioxidant activity of nordihydroguaiaretic acid (NDGA) and ascorbic acid were assessed using a yeast strain that does not grow under hyperoxic conditions. At the concentrations assayed, NDGA did not remediate this sensitivity to hyperoxia, whereas ascorbic acid did. These and additional assays are further reviewed with an aim to collate and standardize secondary assays and streamline antifungal mode of action determinations