Drug Leads from Endophytic Fungi: Lessons Learned via Scaled Production

Abstract

Recently, the isolation and elucidation of a series of polyhydroxyanthraquinones were reported from an organic extract of a solid phase culture of an endophytic fungus, Penicillium restrictum (strain G85). One of these compounds, ω-hydroxyemodin (1: ), showed promising quorum-sensing inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) in both in vitro and in vivo models. The initial supply of 1: was 19 mg, and this amount needed to be scaled by a factor of 30 to 50 times, in order to generate material for further in vivo studies. To do so, improvements were implemented to enhance both the fermentation of the fungal culture and the isolation of this compound, with the target of generating > 800 mg of study materials in a period of 13 wk. Valuable insights, both regarding chemistry and mycology, were gained during the targeted production of 1: on the laboratory-scale. In addition, methods were modified to make the process more environmentally friendly by judicious choice of solvents, implementing procedures for solvent recycling, and minimizing the use of halogenated solvents.

Fig. 1

Structure of the polyhydroxyanthraquinone, ω-hydroxyemodin (1).

Fig. 2

a Left:Penicillium restrictum (G85) at the time of the initial study in 2014, showing red guttates. Right: P. restrictum in 2016 using the same media conditions. Potato dextrose agar (A) Malt extract agar (B) Yeast extract soy peptone dextrose agar (C). Note how the red guttates, a key observation in the initial study [2], were no longer present after repeated transferring and culturing of P. restrictum. In our experience, this sort of ‘domestication’ of a fungus is particularly problematic with endophytic fungi; others have noted this as well [9,21,22]. Fig. 2b When Penicillium restrictum (G85) was grown on SD Agar, the fungus produced a red color that diffused into the media; this correlated well with the production of ω-hydroxyemodin, as well as other polyhydroxyanthraquinones. Fig. 2c Surface sampling of the Penicillium restrictum strain G85 using the droplet probe [16]. A scanned image of P. restrictum on SD Agar. The intensity of the golden color of the dots indicates the relative concentration of ω-hydroxyemodin observed at that position on the plate. Via heat mapping experiments with the droplet probe, ω-hydroxyemodin (1) was shown to be present predominantly in the highly colored areas of the media.

Fig. 3

Schematic of how fermentations of Penicillium restrictum (strain G85) were modified and scaled to produce nearly a gram of ω-hydroxyemodin (1). From start to finish, it took a team of three people approximately 13 weeks to go from <20 mg to >800 mg of 1.

Fig. 4

Schematic of the benefits obtained with the improved method. Benefits at each stage of processing are shown as a summary at the bottom.