Antifungal Drug Synergy; from Proteomics to a Mechanism of Action

Yeast pathogens cause human diseases that range from superficial and largely cosmetic to severe, disseminated life-threatening infections, and can be very difficult to treat. Azole-based antifungal drugs are a mainstay of therapy, particularly in resource-poor regions of the world, but not all infections respond and resistance is an increasing problem.  Drug synergy is one way of improving treatment as it can enhance efficacy and reduce the induction of resistance.  To find targets that might work synergistically with fluconazole (FLC), we subjected Cryptococcus gattii to sub-lethal FLC treatment and analysed the proteomic response over time. We found up-regulation of farnesyl pyrophosphate synthase (FPPS), an enzyme involved in the synthesis of squalene, which feeds into the ergosterol biosynthesis pathway that is subsequently targeted by FLC. FPPS can be inhibited by bisphosphonate drugs, including alendronate (ALN) and zolendronate (ZOL), that are marketed to treat osteoporosis.  ZOL was synergistic with FLC across a range of yeast and mould pathogens. Mechanistic studies revealed synergy was mediated by squalene deprivation, resulting in membrane hyperfluidity and depolarisation, and in the case of Candida glabrata, a complete elimination of the efflux pump activity that largely mediates FLC resistance. We modified ZOL by adding a 10-carbon alkyl tail and found a significant improvement in activity against a remarkably wide spectrum of fungi with low toxicity to human cells.  This study demonstrates the power of ‘omics data to find new synergistic targets that can then be developed into powerful new antifungal therapies.