Alkylation-based optimization of antifungal FPPS inhibitors yields a potent, broad-spectrum lipophilic zoledronate derivative

Farnesyl pyrophosphate synthetase (FPPS) is a critical enzyme for squalene biosynthesis. In fungi, squalene is the primary substrate of the ergosterol biosynthesis pathway, which is the target of azole antifungals. Bisphosphonate-based FPPS inhibitors like zoledronate (ZOL) have limited antifungal activity alone but are highly synergistic with azoles against a broad array of human fungal pathogens. Various bisphosphonate drugs are available and are FDA-approved for bone resorption disorders, but their use as antifungal therapeutics is limited by their poor systemic bioavailability. To overcome this, we synthesized a suite of different FPPS inhibitors that incorporated alkyl tails of various lengths to improve solubility, cell penetration, and FPPS inhibition. A derivative of ZOL with an attached 10-carbon alkyl tail (here named 10-ZOL) was also produced. Most of the inhibitors demonstrated limited efficacy and had inconsistent interactions with azole antifungals; however, 10-ZOL had dramatically improved antifungal activity in vitro across a broad spectrum of pathogens (minimum inhibitory concentration range = 0.5–8 µg/mL), in vivo efficacy in an invertebrate model of infection, and a low level of toxicity in mammalian cells (IC₅₀ = 35.77 µg/mL). Rescue with exogenous squalene revealed that 10-ZOL, like ZOL, acts by inhibiting squalene synthesis, resulting in changes to the membrane lipid environment. Cell-based assays showed that these lipid changes compromised plasma membrane structure and integrity and caused an accumulation of toxic oxygen radicals. We conclude that 10-ZOL is a promising new therapeutic lead due to its broad spectrum of activity and synergistic interactions with azoles.