Bisphosphonates synergistically enhance the antifungal activity of azoles in dermatophytes and other pathogenic molds

Abstract

Superficial infections of the skin, hair, and nails by fungal dermatophytes are the most prevalent of human mycoses, and many infections are refractory to treatment. As current treatment options are limited, recent research has explored drug synergy with azoles for dermatophytoses. Bisphosphonates, which are approved to treat osteoporosis, can synergistically enhance the activity of azoles in diverse yeast pathogens but their activity has not been explored in dermatophytes or other molds. Market bisphosphonates risedronate, alendronate, and zoledronate (ZOL) were evaluated for antifungal efficacy and synergy with three azole antifungals: fluconazole (FLC), itraconazole (ITR), and ketoconazole (KET). ZOL was the most active bisphosphonate tested, displaying moderate activity against nine dermatophyte species (MIC range 64-256 µg/mL), and was synergistic with KET in eight of these species. ZOL was also able to synergistically improve the anti-biofilm activity of KET and combining KET and ZOL prevented the development of antifungal resistance. Rescue assays in Trichophyton rubrum revealed that the inhibitory effects of ZOL alone and in combination with KET were due to the inhibition of squalene synthesis. Fluorescence microscopy using membrane- and ROS-sensitive probes demonstrated that ZOL and KET:ZOL compromised membrane structure and induced oxidative stress. Antifungal activity and synergy between bisphosphonates and azoles were also observed in other clinically relevant molds, including species of Aspergillus and Mucor. These findings indicate that repurposing bisphosphonates as antifungals is a promising strategy for revitalising certain azoles as topical antifungals, and that this combination could be fast-tracked for investigation in clinical trials.

Importance: Fungal infections of the skin, hair, and nails, generally grouped together as "tineas" are the most prevalent infectious diseases globally. These infections, caused by fungal species known as dermatophytes, are generally superficial, but can in some cases become aggressive. They are also notoriously difficult to resolve, with few effective treatments and rising levels of drug resistance. Here, we report a potential new treatment that combines azole antifungals with bisphosphonates. Bisphosphonates are approved for the treatment of low bone density diseases, and in fungi they inhibit the biosynthesis of the cell membrane, which is also the target of azoles. Combinations were synergistic across the dermatophyte species and prevented the development of resistance. We extended the study to molds that cause invasive disease, finding synergy in some problematic species. We suggest bisphosphonates could be repurposed as synergents for tinea treatment, and that this combination could be fast-tracked for use in clinical therapy.

Keywords: Trichophyton; antifungal agents; antifungal therapy; azole; bisphosphonate; dermatophytes; drug synergy; ketoconazole; zolendronate.

Fig 1

Combining ketoconazole and zoledronate prevents the development of antifungal resistance in T. rubrum. (A and B) Trichophyton rubrum R-218 was cultured in 0.25× MIC or MIC_c of KET (1 µg/mL), ZOL (32 µg/mL), and KET:ZOL (0.016:2 µg/mL) in agar and then serially sub-cultured onto solid media containing doubling concentrations of each agent. After 4 days of growth, a photograph was taken (A) and the annular radius of each colony was measured (B). Boxplots represent the mean annular radius of five colonies in each of three independent biological replicate experiments.

Fig 2

Bisphosphonate-azole combinations inhibit dermatophytes by depriving cells of squalene, permeabilizing the membrane and causing oxidative stress. (A) Trichophyton rubrum R-218 was treated with KET at MIC_c (0.25 µg/mL), ZOL at MIC (128 µg/mL), KET:ZOL combined at MIC_c (0.25:8 µg/mL), and a no-drug control (1% DMSO) supplemented with increasing concentrations of squalene. Data are normalized to the no-drug control and inoculum-free media and are the mean of three biological replicates ± SD. (B) Germinating T. rubrum conidia were treated with the no-drug control (1% DMSO), KET at MIC (4 µg/mL), ZOL at MIC (128 µg/mL), and KET:ZOL at MIC_c (0.25:8 µg/mL), stained with DiBAC₄(3) and imaged by bright-field (left) and fluorescence microscopy with a FITC filter (right). (C and D) Germinating conidia were treated with the no-drug control (1% DMSO), KET at MIC (4 µg/mL), ZOL at MIC (128 µg/mL), and KET:ZOL at 1× MIC_c (0.25:8 µg/mL), KET:ZOL at 4× MIC_c (1:32 µg/mL), or H₂O₂ at 0.5× MIC (0.345 mM) and stained with DCFDA to visualize intracellular ROS. Total corrected cell fluorescence was calculated for 50 hyphae in each treatment (C), and hyphae were imaged by bright-field microscopy (D; left) and fluorescence microscopy with a FITC filter (D; right). Bars in (C) indicate the mean corrected cell fluorescence ± SD.