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. 2013 Feb;12(2):278-87.
doi: 10.1128/EC.00314-12. Epub 2012 Dec 14.

A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis

Arielle Butts  1 , Louis DiDone, Kristy Koselny, Bonnie K Baxter, Yeissa Chabrier-Rosello, Melanie Wellington, Damian J Krysan
Affiliations

A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis

Arielle Butts et al. Eukaryot Cell. 2013 Feb.

Abstract

New, more accessible therapies for cryptococcosis represent an unmet clinical need of global importance. We took a repurposing approach to identify previously developed drugs with fungicidal activity toward Cryptococcus neoformans, using a high-throughput screening assay designed to detect drugs that directly kill fungi. From a set of 1,120 off-patent medications and bioactive molecules, we identified 31 drugs/molecules with fungicidal activity, including 15 drugs for which direct antifungal activity had not previously been reported. A significant portion of the drugs are orally bioavailable and cross the blood-brain barrier, features key to the development of a widely applicable anticryptococcal agent. Structural analysis of this set revealed a common chemotype consisting of a hydrophobic moiety linked to a basic amine, features that are common to drugs that cross the blood-brain barrier and access the phagolysosome, two important niches of C. neoformans. Consistent with their fungicidal activity, the set contains eight drugs that are either additive or synergistic in combination with fluconazole. Importantly, we identified two drugs, amiodarone and thioridazine, with activity against intraphagocytic C. neoformans. Finally, the set of drugs is also enriched for molecules that inhibit calmodulin, and we have confirmed that seven drugs directly bind C. neoformans calmodulin, providing a molecular target that may contribute to the mechanism of antifungal activity. Taken together, these studies provide a foundation for the optimization of the antifungal properties of a set of pharmacologically attractive scaffolds for the development of novel anticryptococcal therapies.

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Figures

Fig 1
Fig 1
Representative primary screening data. The scatterplot shows the fold changes in library drug-treated wells relative to DMSO (1%)-treated wells for a representative plate in the primary AK screen.
Fig 2
Fig 2
Representative results of secondary screening assays. (A) Dose-response data for amiodarone (AMD) showing relative light units of released AK activity at the indicated doses relative to those in DMSO (1%) (mock)-treated wells. (B) Percentages of cells staining with propidium iodide relative to DMSO-treated cells, as determined by fluorescence microscopy, for cells treated with amiodarone (AMD), trifluoperazine (TFP), and thioridazine (THZ) at the indicated drug concentrations (n, >100 cells).
Fig 3
Fig 3
Common chemotype of hits. Structural analysis of the hits revealed that a common motif for the set was a hydrophobic/aromatic moiety (blue) linked by a flexible chain (black) to a basic amine (green). The chemical structures of the hits that fit this motif are indicated. Structure numbers correlate with Table 1. The structural features are color coded to indicate the regions that match the chemotype.
Fig 4
Fig 4
Representative interactions of hits with fluconazole. (A) Checkerboard assay showing synergistic interaction of fluconazole with suloctodil. Filled circles indicate growth, and open circles indicate no growth. (B) Subinhibitory concentrations of fluconazole (FLU; 2 μg/ml) and thioridazine (THZ; 16 μg/ml) decreased the initial inoculum (indicated by the horizontal bar) >2 log10 CFU/ml after 24 h.
Fig 5
Fig 5
The set of fungicidal anticryptococcal drugs contains a number of calmodulin antagonists. (A) Thermal shift assay for CnCam1 in the presence of DMSO or the well-characterized calmodulin antagonist triflupromazine (TFP; 100 μM). The x axis shows the temperature, and the y axis shows the negative derivative of the fluorescence value. The red arrow indicates the Tm for CnCam1 in the presence of DMSO, and the green arrow indicates that in the presence of TFP. (B) Changes in Tm induced by the indicated drugs relative to that with DMSO (1%). The bars indicate means for three independent experiments performed in triplicate; error bars indicate standard deviations.
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