RESUMO
Objectives: Over the last 30 years, the number of invasive fungal infections among immunosuppressed patients has increased significantly, while the number of effective systemic antifungal drugs remains low. The aim of this study was to identify and characterize antifungal compounds that inhibit fungus-specific metabolic pathways not conserved in humans. Methods: We screened a diverse compound library for antifungal activity in the pathogenic mould Aspergillus fumigatus . We determined the in vitro activity of bromoquinol by MIC determination against a panel of fungi, bacteria and cell lines. The mode of action of bromoquinol was determined by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring genes and by RNAseq analysis in A. fumigatus . In vivo efficacy was tested in Galleria mellonella and murine models of A. fumigatus infection. Results: Screening of a diverse chemical library identified three compounds interfering with fungal iron utilization. The most potent, bromoquinol, shows potent wide-spectrum antifungal activity that was blocked in the presence of exogenous iron. Mode-of-action analysis revealed that overexpression of the dba secondary metabolite cluster gene dbaD , encoding a metabolite transporter, confers bromoquinol resistance in A. nidulans , possibly by efflux. RNAseq analysis and subsequent experimental validation revealed that bromoquinol induces oxidative stress and apoptosis in A. fumigatus . Bromoquinol significantly reduced mortality rates of G. mellonella infected with A. fumigatus , but was ineffective in a murine model of infection. Conclusions: Bromoquinol is a promising antifungal candidate with a unique mode of action. Its activity is potentiated by iron starvation, as occurs during in vivo growth.
Assuntos
Antifúngicos/farmacologia , Apoptose , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus nidulans/efeitos dos fármacos , Estresse Oxidativo , Quinolinas/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Modelos Animais de Doenças , Lepidópteros , Testes de Sensibilidade Microbiana , Análise de SobrevidaRESUMO
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.
Assuntos
Antifúngicos/farmacologia , Heme/antagonistas & inibidores , Heme/biossíntese , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Animais , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Linhagem Celular , Farmacorresistência Fúngica , Sinergismo Farmacológico , Ferroquelatase/antagonistas & inibidores , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Humanos , Insetos , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/microbiologia , Protoporfirinas/biossínteseRESUMO
Invasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans sensitive to cell wall-active agents. Eight "hit" compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-chloro-6-arylamino-7-nitro-benzofurazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic Aspergillus, Candida, Fusarium, and Rhizopus isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with Aspergillus nidulans and Saccharomyces cerevisiae indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (Galleria mellonella, Drosophila melanogaster) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.