Emestrins, anti-Cryptococcus epipolythiodiketopiperazines from Podospora australis

Cryptococcus species are among the most common causes of invasive fungal infections worldwide, affecting one million people each year, and resulting in >600,000 deaths. Current treatments employ a limited number of antifungal agents (amphotericin B, flucytosine, fluconazole) which remain unsatisfactory because of their toxicity and the emergence of drug resistance. Very few antifungal agents are currently in clinical development, none of which have been chosen based on effectiveness against Cryptococcus. Natural products (NPs) from fungi have been critical to the development of antifungal agents, e.g., echinocandins. However, discovery of anti-Cryptococcus NPs generally has been a by-product of Candida-driven screening. On the basis of these considerations, we hypothesized that screening underinvestigated types of fungi for active NPs using a Cryptococcus-centric approach would offer outstanding prospects for discovery of new antifungal agents. Thus, we have undertaken a project focused on exploring fermentation extracts of rarely-studied coprophilous (dung-colonizing) fungi for NPs with activity against C. neoformans. In the course of this work, two strains of Podospora australis (Ascomycota, Lasiosphaeriaceae) were isolated from horse and rabbit dung. Their culture extracts strongly inhibited growth of C. neoformans, but negligibly affected Candida albicans. Bioassay-guided fractionation of these extracts led to the isolation of twelve emestrin-type epipolythiodiketopiperazine metabolites, including four new compounds, which we named emestrins H–K. Their structures were elucidated by analysis of NMR and MS data. Four of the emestrins encountered showed significant anti-Cryptococcus activity. Here we report details of the production, isolation, and structure elucidation of these compounds, and their bioactivity against C. neoformans.