S3.4c A pipeline toward the identification of novel antifungal compounds derived from the microbial dark matter

Abstract

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PM Background: The current armamentarium of antifungal drugs and the restricted variety in antifungal drug classes combined with the ever-rising threat of resistant fungal pathogens highlighted the urgent need for novel antifungal compounds. Natural antifungal secondary metabolites have always been the prevalent source for drug development, exemplified by the echinocandin and polyene drug classes. Yet, the golden age discovery platforms were abandoned due to compound rediscovery and its paired economic cost. Study: In an effort to revive the original success stories, we combined the traditional approach of sampling and screening for active secondary metabolites with present-day advances in sequencing, genome mining, impedance spectroscopy, HPLC, LCMS, and NMR. Soil bacteria and fungi were isolated through in situ cultivation via the iChip method. After application of the OSMAC approach, 389 broths were identified with activity against Candida albicans. To prioritize active extracts, several criteria were set up; low to absent mammalian host cell toxicity, activity against a broad spectrum of fungal pathogens including wild-type reference strains, and established antifungal drug-resistant variants and species identification of the producing strain. Continuing, Lead hits were purified utilizing bioactivity-based semi-preparative HPLC. The resulting pure fractions were analyzed by tandem LCMS-MS, and proposed structures were later confirmed with NMR. In vitro and in vivo validation of the purified compounds will be performed. Additionally, aside from discovering a novel antifungal compound, another project goal is to gauge if impedance spectroscopy can provide an early suggestion regarding the mode of action of the present antifungal agent. For this, a POC study was performed which showed that different antifungal drug classes provide distinct signature response profiles by which they can be classified. As such, when active secretion broths show unique impedance profiles, in comparison with the signature profiles of established antifungal drugs, it suggests that they work through a different mode of action. Results Several species were identified as producing antifungal secondary metabolites that are currently absent in the literature. Either the compound itself was unknown or literature never described the species as a producer of a known, or variant of a known antimycotic compound. Moreover, several species are novel based on Illumina sequencing. Genera producing our current lead hits include bacteria: Pseudomonas, Tsukamurella, Paraburkholderia, and fungi: Athelia, Penicillium. Within the collection, the Pseudomonas species appear to produce variants of the antimycotic iron-chelating pyoverdine class.