Fungal infections impact the lives of at least 12 million people every year, killing over 1.5 million. Wide-spread use of fungicides and prophylactic antifungal therapy have driven resistance in many serious fungal pathogens, and there is an urgent need to expand the current antifungal arsenal. Recent research has focused on improving azoles, our most successful class of antifungals, by looking for synergistic interactions with secondary compounds. Synergists can co-operate with azoles by targeting steps in related pathways, or they may act on mechanisms related to resistance such as active efflux or on totally disparate pathways or processes. A variety of sources of potential synergists have been explored, including pre-existing antimicrobials, pharmaceuticals approved for other uses, bioactive natural compounds and phytochemicals, and novel synthetic compounds. Synergy can successfully widen the antifungal spectrum, decrease inhibitory dosages, reduce toxicity, and prevent the development of resistance. This review highlights the diversity of mechanisms that have been exploited for the purposes of azole synergy and demonstrates that synergy remains a promising approach for meeting the urgent need for novel antifungal strategies.

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 targetted farnesyl pryrophosphate synthetase (FPPS), an enzyme in the squalene biosynthesis pathway, using nitrogenous bisphosphonates. We hypothesised 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 (GM MIC = 113.03 mg/L; risedronate = 378.49 mg/L; alendronate = 158.4 for) and was broadly synergistic when combined with fluconazole, with an FICI ≤ 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.

Fungal infections impact the lives of at least 12 million people every year, killing over 1.5 million. Wide-spread use of fungicides and prophylactic antifungal therapy have driven resistance in many serious fungal pathogens, and there is an urgent need to expand the current antifungal ar-senal. Recent research has focused on improving azoles, our most successful class of antifungals, by looking for synergistic interactions with secondary compounds. Synergists can co-operate with azoles by targeting steps in related pathways, or they may act on mechanisms related to re-sistance like active efflux or on totally disparate pathways or processes. A variety of sources of potential synergists have been explored, including pre-existing antimicrobials, pharmaceuticals approved for other uses, bioactive natural compounds and phytochemicals, and novel synthetic compounds. Synergy can successfully widen the antifungal spectrum, decrease inhibitory dosag-es, reduce toxicity, and prevent the development of resistance. This review highlights the diversity of mechanisms that have been exploited for the purposes of azole synergy and demonstrates that synergy remains a promising approach for meeting the urgent need for novel antifungal strate-gies.