Plant-Derived Isoquinoline Alkaloids that Target Ergosterol Biosynthesis Discovered by Using a Novel Antifungal Screening Tool

Background: The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. Methods: In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte,Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. Findings: These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. Interpretation: By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-β-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo. Funding Statement: This work was supported by Faculty Research Grant, Hong Kong Baptist University (FRG 2/17-18/103), the Hong Kong Baptist University (HKBU) Interdisciplinary Research Matching Scheme (RC-IRMS/15-16/02),NSFC funding 31670360, 81973293, U1702286 and Natural Science Foundation of SZU 860-000002110131. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Female guinea pigs were used for the in vivo antifungal study, which was approved by the laboratory animal ethics committee of Shenzhen University, China [Approval number: SYXK (Yue) 2014-0140].