Inhibition of Candida species via Proteosome Inhibitor MG-262 (ZL3B)

Abstract

Backround: Candida species take the fourth place among the microorganisms which cause hospital infections. New therapeutic agents are needed because of rapidly increasing resistance rates. Boric acid preparations are preferred in local treatment especially in resistant cases while azole formulations are the first choice in oral treatment. The only boric acid analog that was approved by FDA (Food and Drug Administration) for systemic use is Bortezomib (Velcade). Bortezomib is a proteasome inhibitor and proteasome inhibitors cause apoptosis of eukaryotic cells. Although the yeast also has a well preserved 20S proteasome region, the studies on the effects of proteasome inhibitors on the yeast are limited. In this study, the effects of the one of the most potent proteasome inhibitor, MG-262, on Candida spp. were investigated. Methods: C. albicans ATCC 90028, C. krusei ATCC 6258 and C. glabrata (clinical strain) strains were used in our study. Antifungal susceptibilities of the strains against MG-262 and effects of MG-262 on oxidative metabolism, proteasome activity and apoptosis as well as virulence factors such as hyphal transformation, pseudohypha, germ tube and biofilm formation were investigated. CLSI (Clinical and Laboratory Standards Institute) guidelines were used for antifungal susceptibility tests. The effect on hypha and pseudohypha formation and germ tube were determined with conventional tests. Biofilm formation was detected according to the microplate method. Oxidative metabolism experiments, proteosome activity and apoptosis tests were performed with Alamarblue dye, fluorogenic peptides and DNA ladder assays, respectively. Findings: MG-262 exhibited minimum inhibitory activity at 25 μg/ml concentration for C. albicans ATCC 90028 and at 50 μg/ml concentration for C. krusei ATCC 6258 and C. glabrata. Minimum fungicidal concentrations for all species were 50 μg/ml. Hypha, pseudohypha, germ tube and biofilm formation, proteosome activity and oxidative metabolism were all inhibited at 50 μg/ml and higher concentrations. The apoptosis was detected at 200 μg/ml. Conclusion: MG-262 caused apoptosis by inhibiting oxidative metabolism and showed fungicidal activity. It also inhibited the virulence factors such as hypha, pseudohypha germ tube and biofilm formation. Inhibition of proteasome is promising in terms of discovery of a new generation antifungal agent.