Amino-acid derived 1,2 benzisothiazolinone derivatives as novel small molecule antifungal inhibitors: Characterization and identification of potential genetic targets

Abstract

A steady increase in the incidence of fungal infections has been observed over the past few decades, and treatment remains challenging especially for immuno-compromised populations. Identification of the ideal therapeutic agent to treat fungal infections still remains elusive. Such an agent would need to have broad antifungal activity, low rates of resistance and minimal adverse effects. Since none of the current drugs fulfill the criteria for an ideal agent, it is imperative to identify novel drug candidates. In the course of screening for antifungal compounds, a scaffold of synthetic compounds with antifungal activity was discovered. These compounds have no structural similarity to any existing antifungal compound. 4 amino-acid derived 1,2 benzisothiazolinone derivatives (BD-I-132/DFD-VI-15, BD-I-186, DFD-V-49 and DFD-V-66) show anti-fungal activity against Candida sp., Aspergillus fumigatus, Cryptococcus neoformans and dermatophytes at MIC-50 concentrations in the range of 0.8-12.5 microg/ml. All compounds were fungicidal against Candida sp. The compounds also maintained their MIC-50 values against azole, polyene and echinocandin-resistant strains of Candida sp. The MICs of the compounds were also unaffected by strains that over-expressed drug efflux pumps. A genetics based approach which involves the use of a homozygous deletion collection of approximately 4700 Saccharomyces cerevisiae mutants was used to identify potential gene targets of DFD-VI-15. All mutants were treated with a) suboptimal levels of compound to identify hypersensitive strains and b) fungicidal concentrations of compound to identify resistant strains. 32 mutants were found to be hypersensitive compared to parent strain, BY4743. Also, 96 mutants showed resistance to this compound. Both clusters identified mutants that were essential to mitochondrial function. Saccharomyces and Candida strains (BY4743 and CAF2) were grown in the absence of oxygen and found to be insensitive to the compound. Also, both strains were hypersensitive to DFD-VI-15 when grown on a non-fermentable carbon source like glycerol. Flowcytometry experiments show that DFD-VI-15 treated Candida strains have higher ROS levels when compared to non-treated strains. This suggests that the DFD-VI-15 exerts its antifungal effect by affecting mitochondrial function.