The Antifungal and Synergistic Effect of Bisphosphonates in Cryptococcus

New treatment strategies are required for cryptococcosis, a leading mycosis in HIV-AIDS patients. Following the identification of Cryptococcus proteins differentially expressed in response to fluconazole, we targeted farnesyl pryrophosphate synthetase (FPPS), an enzyme in the squalene biosynthesis pathway, using nitrogenous bisphosphonates. We hypothesized that these would disrupt squalene synthesis and thereby produce synergy with fluconazole, which acts on a downstream pathway that requires squalene. The susceptibilities of 39 clinical isolates from 6 different species of Cryptococcus were assessed for bisphosphonates and fluconazole, used both independently and in combination. Effective fluconazole-bisphosphonate combinations were then assessed for fungicidal activity, efficacy against biofilms, and ability to resolve cryptococcosis in an invertebrate model. The nitrogenous bisphosphonates risedronate, alendronate, and zoledronate were antifungal against all strains tested. Zoledronate was the most effective (geometric mean MIC = 113.03 mg/liter; risedronate = 378.49 mg/liter; alendronate = 158.4 mg/liter) and was broadly synergistic when combined with fluconazole, with a fractional inhibitory concentration index (FICI) of ≤0.5 in 92% of isolates. Fluconazole and zoledronate in combination were fungicidal in a time-kill assay, inhibited Cryptococcus biofilms, prevented the development of fluconazole resistance, and resolved infection in a nematode model. Supplementation with squalene eliminated bisphosphonate-mediated synergy, demonstrating that synergy was due to the inhibition of squalene biosynthesis. This study demonstrates the utility of targeting squalene synthesis for improving the efficacy of azole-based antifungal drugs and suggests bisphosphonates are promising lead compounds for further antifungal development.