Identification and characterization of haemofungin, a novel antifungal compound that inhibits the final step of haem biosynthesis

Abstract

Objectives: During recent decades, the number of invasive fungal infections among immunosuppressed patients has increased significantly, whereas the number of effective systemic antifungal drugs remains low and unsatisfactory. The aim of this study was to characterize a novel antifungal compound, CW-8/haemofungin, which we previously identified in a screen for compounds affecting fungal cell wall integrity.

Methods: The in vitro characteristics of haemofungin were investigated by MIC evaluation against a panel of pathogenic and non-pathogenic fungi, bacteria and mammalian cells in culture. Haemofungin mode-of-action studies were performed by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring plasmids and biochemical validation of the target. In vivo efficacy was tested in the Galleria mellonella and Drosophila melanogaster insect models of infection.

Results: We demonstrate that haemofungin causes swelling and lysis of growing fungal cells. It inhibits the growth of pathogenic Aspergillus, Candida, Fusarium and Rhizopus isolates at micromolar concentrations, while only weakly affecting the growth of mammalian cell lines. Genetic and biochemical analyses in A. nidulans and Aspergillus fumigatus indicate that haemofungin primarily inhibits ferrochelatase (HemH), the last enzyme in the haem biosynthetic pathway. Haemofungin was non-toxic and significantly reduced mortality rates of G. mellonella and D. melanogaster infected with A. fumigatus and Rhizopus oryzae, respectively.

Conclusions: Further development and in vivo validation of haemofungin is warranted.

Figure 1

Haemofungin causes morphological changes characteristic of damage to the cell wall of A. fumigatus. (a) Molecular structure of haemofungin, a benzimidazole derivative. (b–d) Freshly harvested spores of A. fumigatus strain Af293 were incubated for 24 h in the presence of 0.8 μM haemofungin and analysed microscopically by light, fluorescence or TEM microscopy, CFW cell wall staining or DiBAC staining of dead cells. Cell swelling and lysis [arrow, (b), light microscopy], abnormal cell wall morphology (TEM) and increased CFW staining of wall polysaccharides reveal haemofungin-induced wall damage. DiBAC staining indicates partial cell death in discrete areas of the hyphae. Black bar = 10 μm.

Figure 2

Mapping and identification of the A. nidulans AN7752 gene encoding HemH/ferrochelatase that confers haemofungin resistance upon overexpression. (a) Schematic representation of the inserts in each of the three resistance-conferring high-copy plasmids. Only the gene AN7752 is shared by all three plasmids. (b) Amino acid similarity between AN7752 and homologues of select fungal, human and Escherichia coli ferrochelatase proteins according to pBLAST.

Figure 3

Haemofungin induces the expression of key haem biosynthetic genes in A. fumigatus. A. fumigatus overnight culture grown in liquid medium (either CM or MM) was treated with 5 μM haemofungin for 1 h. Total RNA was extracted and analysed by northern blotting with probes specific for the haem biosynthetic genes hemA, hemB, hemE, hemF and hemH. The expression of all genes except hemB was increased in the presence of haemofungin. Bottom panel: total RNA loading control.

Figure 4

Treatment with haemofungin causes accumulation of PPIX in A. fumigatus. A. fumigatus overnight culture grown in YAG liquid medium was treated with 2 μM haemofungin for 2 h. The mycelium was (a) spun down and directly viewed for accumulation of porphyrins and (b) lyophilized and analysed for PPIX content by HPLC (C18) column separation.

Figure 5

Toxicity and efficacy analysis of haemofungin. For analysis of toxicity, G. mellonella larvae (a) were injected once with up to 22.7 mg/kg haemofungin and Drosophila Tl^−/− flies (c) were fed with fly food containing up to 14.4 mg/mL haemofungin for 7 days. For analysis of survival, G. mellonella larvae (b) and Drosophila Tl^−/− flies (d) were infected with A. fumigatus (Af293) or R. oryzae conidia and treated with up to 5.7 mg/kg and 14.4 mg/mL haemofungin, respectively. Treatment with amphotericin B was used as a positive control in G. mellonella.