Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo

Abstract

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.

Importance: Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Figures

FIG 1

Anti-cryptococcal activity of triphenylethylenes. (A) MICs of the indicated molecules against C. neoformans var. grubii strain H99 as determined by CLSI standard methods. CLM, clomiphene; TAM, tamoxifen; TOR, toremifene; 4-HO-TAM, 4-hydroxy-tamoxifen; ENDO, endoxifen; IDX, idoxifene; M-IDX, methylene-idoxifene. The chemical structures for each molecule are shown in Fig. S1A. The y axis of the graph is a modified log2 scale. (B) The combination of subinhibitory concentrations of toremifene (TOR; 2 µg/ml) and fluconazole (FLU; 2 µg/ml) reduces the initial inoculum of C. neoformans (4 log10 CFU/ml) more than 2 log10 CFU/ml at 24 h and is therefore a fungicidal combination. (C) J774 mouse macrophages were infected with opsonized C. neoformans, washed to remove nonadherent/nonphagocytosed cells, and incubated with fresh medium containing either DMSO (1%) or drug for 24 h. After 24 h, macrophages were lysed and the number of C. neoformans CFU per well determined as described in Materials and Methods. Data are means of triplicates, and error bars represent standard deviations. Differences between groups were statistically significant (Student’s t test; P < 0.05). (D) Male A/JCr mice (7 per group) were infected with serotype A C. neoformans H99 by tail vein injection. The control group (CTRL) received saline by intraperitoneal injection and peanut oil by oral gavage. The fluconazole group (FLU) received 5 mg/kg FLU by intraperitoneal injection and peanut oil by oral gavage. The tamoxifen group (TAM) received saline by intraperitoneal injection and 200 mg/kg tamoxifen suspended in peanut oil by oral gavage. The combination treatment group (TAM+FLU) received 5 mg/kg fluconazole by intraperitoneal injection and 200 mg/kg tamoxifen suspended in peanut oil by oral gavage. The bars represent the median for each group, and each dot represents an individual animal. The number of CFU/gram of brain tissue was determined and transformed into log10 units, and differences between groups were analyzed by ANOVA; statistical significance was set at a P value of <0.05 (SigmaPlot software).

FIG 2

Toremifene binds and inhibits the function of C. neoformans calmodulin in vitro. (A) C. neoformans strain (cam1Ts) with reduced CAM1 expression (38) was transformed with empty vector, a vector expressing CAM1 from its endogenous promoter (pCAM1), or a vector expressing CAM1 from the strong constitutive promoter GPD1 (pGPD-CAM1). Serial dilutions (10-fold) of a suspension (1 OD600 unit/ml) suspension of cells for the indicated strains were spotted on yeast peptone dextrose (YPD) plates containing DMSO solvent (1%) or toremifene (TOR; 8 µg/ml). The plates were incubated at 30°C for 3 days and photographed. (B) Thermal denaturation curves for CnCam1 in the presence of DMSO (green), trifluopromazine (TFP; red), and toremifene (TOR; blue), as determined by differential scanning fluorimetry. The y axis shows the negative derivative of the fluorescence with respect to change in temperature. The arrows indicate low-temperature and high-temperature inflection points corresponding to the Tm for N-terminal-domain and C-terminal-domain denaturation, respectively. (C) CnCam1-mediated activation of human calcineurin is inhibited by TOR. The specific activity (absorbance [abs]/mol calcineurin/min) of human calcineurin is plotted on the y axis, and the no-drug (DMSO-only) control indicates activation. The dose-response curve was fitted by linear analysis (R = 0.986) to give an IC50 of 25 µg/ml. Three independent experiments were performed with similar results, and results of a representative single experiment are shown. Error bars indicate the standard deviations for technical replicates of each drug concentration.

FIG 3

Toremifene phenocopies vacuolar defects of calmodulin mutants and inhibits stress-induced nuclear localization of Crz1. (A) Logarithmic-phase C. neoformans H99 cells were treated with subinhibitory toremifene (2 µg/ml) and the calmodulin antagonist fluspirilene (32 µg/ml), stained with the vacuolar marker MDY-64, and photographed in bright-field or fluorescence channels. The arrowhead indicates a normal, moderate-sized vacuole in DMSO-treated cells, and arrows indicate fragmented, small vacuoles indicative of vacuole fusion defects. (B) C. neoformans strains containing fluorescently tagged Nop1-GFP and Crz1-mCherry were cultivated to logarithmic phase at 30°C, 37°C, and 37°C with a subinhibitory concentration of toremifene (2 µg/ml). Photomicrographs from the appropriate channels are shown, as well as a merged fluorescence image. (C) The percentage of cells with colocalized Nop1-GFP and Crz1-mCherry under the indicated conditions is graphed. Cells were also treated with subinhibitory concentrations of the known calmodulin inhibitor trifluopromazine (8 µg/ml; 1/4 MIC) as a positive control. Bars indicate means from three independent experiments (>100 cells counted per condition per experiment), and error bars indicate standard deviations.

FIG 4

Toremifene binds the EF hand protein Cml1 and modulates its ability to activate calcineurin. (A) Serial dilutions (10-fold) of suspensions (1 OD600 unit/ml) of cells for wild-type (CM018) and cml1Δ strains were spotted on yeast-peptone-dextrose (YPD) plates containing DMSO solvent (1%) or toremifene (TOR; 8 µg/ml). The plates were incubated at 30°C for 3 days and photographed. (B) TOR (blue trace) shifts the Tm for denaturation of Cml1 compared to solvent control (DMSO, red trace). Differential scanning fluorimetry was performed as described in the legend to Fig. 2 and in Materials and Methods. Arrows indicate the Tm for the two conditions, which were determined as described in Materials and Methods. (C) TOR increases the activity of Cml1-mediated calcineurin activity. The specific activity (absorbance [abs]/mol calcineurin/min) of human calcineurin is plotted on the y axis, and the no-drug (DMSO-only) control indicates activation.

FIG 5

Anti-cryptococcal activity of triphenylethylenes correlates with calmodulin antagonism. C. neoformans strain containing fluorescently tagged Nop1-GFP and Crz1-mCherry were cultivated to logarithmic phase at 37°C with the indicated concentrations of toremifene (TOR), idoxifene, and methylene-idoxifene. As in Fig. 3B, the percentage of cells with colocalized Nop1-GFP and Crz1-mCherry under the indicated conditions is graphed. Bars show the means from three independent experiments (>100 cells counted per condition per experiment), and error bars indicate standard deviations.