The Study Of Antifungal Activities And Underlying Mechanisms Of Thiosemicarbazone Derivatives

Invasive fungal infections are a leading cause of morbidity and mortality in immunocompromised individuals,and the increase in the immunocompromised patient population in recent decades has been accompanied by a rise in the incidence of clinically relevant infections.Despite the substantial societal and financial burdens posed by fungal infections,few antifungals are available,particularly for the treatment of invasive fungal infections.Currently used antifungal drugs mainly fall into three major classes:polyenes,azoles and echinocandins.While echinocandins have a great safety profile,other conventional antifungals,such as amphotericin B,voriconazole,and itraconazole,possess severe toxicity.The rapidly evolving drug-resistance in pathogenic fungi and the slow development of new therapeutics have brought severe challenges to the global health system.New antifungal agents with novel mode of actions are urgently needed to deal with this serious situation.A critical problem in developing antifungals is the potential for cytotoxicity due to the homology of fungal targets with mammalian proteins.The host can hinder microbial growth and virulence through restricting the access of microbes to key micronutrients such as iron and zinc,which is termed as nutritional immunity.This suggests that preventing metal acquisition by fungal pathogens through metal chelation is an alternative approach to copy with fungal infections.As iron chelators,thiosemicarbazone derivatives have played an important role in antifungal effects.However,little is known about their antifungal activities and potential mechanisms.In this study,we systematically evaluated the antifungal activities of these thiosemicarbazone derivatives.Several exhibited broad and potent activity against three common fungal genera:Candida,Cryptococcus,and Aspergillus.19ak,which had the most potent activity against Candida species with the MIC80 value of 0.125 μg/mL,and minimal cytotoxicity against mammalian cells,was chosen for further investigation.Besides,19ak significantly improved the survival of C.albicans-infected Galleria mellonella.In order to further explore how thiosemicarbazone compounds play an antifungal role by chelating ferrous ions and zinc ions and the downstream effect of 19ak chelated ferrous ions and zinc ions on fungal cell function,we performed RNA-seq analysis on C.albicans SC5314.Through GO analysis and KEGG analysis of differential genes,we have elucidated the potential antifungal mechanism of 19ak.Mechanism investigations revealed that 19ak inhibited fungal mitochondrial respiration mainly by retarding mitochondrial respiratory chain complex I activity through iron chelation,and further reduced mitochondrial membrane potential and intracellular ATP synthesis.Moreover,19ak inhibited fungal ribosome biogenesis mainly by disrupting intracellular zinc homeostasis.19ak also stimulated the activities of antioxidant enzymes and decreased reactive oxygen species formation in fungal cells,resulting in an increase in detrimental intracellular reductive stress.However,we did not observe notable depletion of ferrous and zinc ion by 19ak in our tested mammalian cells and the respiratory activity of mammalian cells was also not distinctly affected.19ak exhibited low capacity to induce drug resistance,probably due to a lack of energy to support resistance mechanisms in treated cells.These findings support further potential applications of thiosemicarbazone derivatives in treating fungal infections.