Effect of copper on the antifungal activity of disulfiram (Antabuse^®) in fluconazole-resistant Candida strains

Abstract

Disulfiram (Antabuse®) is an alcohol use disorder medication that exhibits antifungal activity against Candida species. The purpose of this investigation was to determine if copper potentiates the antifungal effects of disulfiram based on prior observations that the combination demonstrates increased antitumor activity. Our findings revealed that copper addition conferred up to an eight-fold reduction in the minimum inhibitory concentrations (MICs) of disulfiram by broth microdilution assessment. Unexpectedly, copper was also found to nullify the fungicidal activity of disulfiram despite the significant reduction in MICs. It was therefore concluded that copper likely increased the antifungal potency of disulfiram through formation of a fungistatic chelation complex.

Lay summary

The effect of copper on the antifungal activity of disulfiram was evaluated against fluconazole-resistant Candida species. The study establishes that copper addition confers greater inhibition of disulfiram-treated Candida cultures, but the combination antagonizes the killing effects of disulfiram.

Introduction

Opportunistic yeasts from the Candida genus are the most frequent cause of systemic mycoses in hospitals. Risk factors for healthcare-associated candidiasis include intravascular or indwelling urinary catheter use, prolonged treatment with broad-spectrum antibiotics, and immunocompromised status due to transplantation or cancer chemotherapy.¹ Candidiasis often develops as an endogenous infection by C. albicans, which is considered susceptible to first-line fluconazole at MICs ≤ 2 μg mg⁻¹.² Fluconazole-resistance is primarily observed in other Candida species, including C. auris, C. glabrata, and intrinsically resistant C. krusei. A significant concern is the global emergence of multidrug-resistant C. auris strains that exhibit MICs above CLSI resistance breakpoints for drugs represented by the three main antifungal classes comprised of the azoles (e.g., fluconazole, FLU), echinocandins (e.g., caspofungin, CAS), and polyenes (i.e., amphotericin B, AMB).³ Accordingly, there is a need for additional mechanistic classes of safe antifungals with coverage against drug-resistant Candida species.

A strategy to accelerate development is to repurpose drugs that are deemed safe by regulatory agencies for human use. The daily maintenance medication disulfiram (Antabuse^®) for alcohol use disorder⁴ is an example of an oral drug that has been assessed in clinical trials for alternative indications including cocaine dependence,⁵ small cell lung cancer,⁶ recurrent glioblastoma,⁷ and latent HIV infections.⁸ In cancer trials, disulfiram (DSF) with copper (Cu²⁺) salt supplements were evaluated due to the enhanced antitumor effects conferred through formation of a cytotoxic, dimeric complex between Cu²⁺ and the cleavage product, diethyldithiocarbamate (DDTC) (Fig. 1).^9–11 These findings prompted us to determine if Cu²⁺ increases the antifungal activity of DSF against Candida species including FLU-resistant C. glabrata and C. auris.^12,13

Figure 1.

Disulfiram forms a dimeric complex on treatment with copper.

Methods

Susceptibility testing

Minimum inhibitory concentrations (MICs) were determined by the microdilution assay method according to Clinical and Laboratory Standards Institute (CLSI) standards in RPMI-1640 medium supplemented with 2% glucose and buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) to pH 7.0.² Stocks of test compounds were prepared in DMSO (DSF, FLU, and AMB) or ultrapure water (DDTC, CAS) prior to testing. Overnight cultures of Candida isolates (Supplementary Tables 1 and 2) diluted to 10³ cells/ml in RPMI were treated with two-fold dilutions of the test compounds in 96-well plates. Following incubation at 35°C in a water-jacketed incubator, the MICs were recorded as the lowest drug concentration that conferred apparent visual growth inhibition after 24 h. Minimum fungicidal concentrations (MFCs) were determined by applying 5 μl of treated inoculums on yeast extract peptone dextrose (YPD) agar to detect for microbial growth after 48-h incubation.

Growth studies

Overnight cultures diluted to 10³ cells/ml in RPMI were used in the following growth studies. Experiments comparing growth by turbidity of 100 μl treated vs. untreated cultures were performed in flat-bottom 96 well plates at 35°C. Optical density (OD) readings were recorded at 530 nm on a Molecular Devices SpectraMax^® 384 plate reader following 10 s agitation every 0.5 h. The 2-h interval absorbances were recorded and plotted using Prism 9.0.2 (GraphPad Software, Inc). Time-kill studies were performed on cultures treated with DSF + Cu²⁺ combinations in 1 ml of RPMI at 35°C with shaking (200 rpm). At time points 0, 8, and 24 h, colony counts were determined from 20 μl of serially diluted samples in saline and grown on YPD agar. The mean cfu/ml for each cohort following 48-h incubation at 35°C is reported.

Flow cytometry studies

An overnight culture of C. glabrata HM-1123 diluted to 10⁶ cells/ml in either RPMI or RPMI with 10 μM CuSO₄ was dispensed (984 μl) in 1.5 ml microfuge tubes containing 16 μl of either 0.5 μg ml⁻¹ DSF or 1 μg ml⁻¹ DDTC. Following incubation (35°C, 18 h, 200 rpm), the tubes were centrifuged (15 000 rpm, 2 min) and the supernatant carefully discarded. The cells were washed twice with PBS (500 μl), resuspended in 1 ml of PBS, and stained with 1 μl of 20 μM propidium iodide (PI) and 3.34 μM SYTO 9 for 0.5 h at 35°C in the dark.¹⁴ Cell viability was then assessed using Novocyte 2000R flow cytometer (ACEA Biosciences, San Diego, CA, USA) equipped with a 488 nm excitation laser and 530/30 (green fluorescence) and 675/30 (red fluorescence) emission filters to detect for SYTO 9 and PI, respectively. Forward scatter (FSC) and side scatter (SSC) plot comparison of unstained cells with PBS was used to calibrate the acquisition gate and minimize background noise. For each sample, ca. 90 000 events were collected and plotted using NovoExpress 1.3.0 software.

Results

Susceptibility testing

Table 1 compares the effect of Cu²⁺ treatment on Candida susceptibility to DSF, DDTC and FLU. The MIC range of DSF was found to be 2 to 4 μg ml⁻¹ (MIC₅₀ 4 μg ml⁻¹) for the ten isolate panel that included FLU-resistant C. albicans (P60002), C. glabrata (AR0317), and C. auris (AR0390) strains. The data also showed that DDTC was a weaker inhibitor of Candida growth than DSF with a panel MIC range of 8 to > 16 μg ml⁻¹ (MIC₅₀ 16 μg ml⁻¹). Further comparisons with Cu²⁺ treated cultures revealed up to an 8-fold reduction in MICs for both agents, while no change was detected for FLU. The panel MIC ranges for DSF + Cu²⁺ and DDTC + Cu²⁺ treated cultures was 0.25 to 1 μg ml⁻¹ (MIC₅₀ 0.5 μg ml⁻¹) and 0.5 to 2 μg ml⁻¹ (MIC₅₀ 1 μg ml⁻¹), respectively.

Table 1.

Effect of 10 μM CuSO₄ on the susceptibility of nine Candida species to DSF, DDTC, and FLU.

		DSFa		DDTCa		FLUa
Species	Strain	-Cu2+	+Cu2+	-Cu2+	+Cu2+	-Cu2+	+Cu2+
C. albicans	P34048	2	0.25	8	0.5	0.5	0.5
C. albicans	P60002	4	0.5	8	0.5	>16	>16
C. auris	AR0390	4	0.5	16	0.5	>16	>16
C. duobushaemulonii	AR0391	2	0.25	16	1	8	8
C. glabrata	AR0317	2	0.5	16	1	>16	>16
C. haemulonii	AR0395	4	0.5	>16	1	4	4
C. kefyr	AR0588	4	1	16	2	1	1
C. lusitaniae	AR0398	4	0.5	16	2	1	1
C. pelliculosa	AR0586	2	0.25	8	1	4	4
C. tropicalis	HM-1124	4	0.25	>16	1	4	4

^aMICs (μg mL^-1) of disulfiram (DSF), diethyldithiocarbamate (DDTC), and fluconazole (FLU).

A more comprehensive assessment of antifungal activity was performed with 30 clinical isolates of C. glabrata and C. auris. Table 2 shows that the addition of Cu²⁺ lowered the MIC₉₀ of DSF from 4 to 1 μg ml⁻¹ against a 20-member C. glabrata panel that included isolates with FKS mutations conferring echinocandin resistance (Supplementary Table 2). A comparable MIC₉₀ value was observed with DDTC + Cu²⁺; however, the effect of Cu²⁺ more pronounced with DDTC. Similar MIC reductions were observed for C. auris with both agents exhibiting a MIC₉₀ of 1 μg ml⁻¹ in Cu²⁺ treated cultures.

Table 2.

Effect of 10 μM copper sulfate on the susceptibility of C. glabrata and C. auris to DSF and DDTC.

	C. glabrata (n = 20)			C. auris (n = 10)
treatmenta	MIC50	MIC90	Range	MIC50	MIC90	Range
DSF	2	4	2–8	4	8	2–8
DDTC	16	16	8–16	16	>16	16 – >16
DSF + Cu2+	1	1	0.5–1	0.5	1	0.25–1
DDTC + Cu2+	1	2	1–2	1	1	0.5–2
FLU	8	128	4–128	128	>256	4 – >256
AMB	0.25	0.5	<0.25–0.5	0.5	4	0.5–4
CAS	0.5	16	<0.25–16	0.5	1	<0.25–16

^aDSF: disulfiram; DDTC: diethyldithiocarbamate; FLU: fluconazole; AMB: amphotericin B; CAS: caspofungin.

Growth studies

The effect of Cu²⁺ on the antifungal activity of DSF and DDTC was further examined by growth curve analysis. Figure 2B shows that 0.25× MIC of DSF (left) and DDTC (right) alone delayed C. glabrata growth for up to 8 h when compared to untreated controls. The addition of 10 μM CuSO₄ extended the growth inhibition to about 12 h in comparison. Despite these findings, the addition of Cu²⁺ to DSF and DDTC unexpectedly reduced fungicidal activity. Figure 2B depicts that 5 μM Cu²⁺ raised the MFC of DSF from 8 μg ml⁻¹ to 16 μg ml⁻¹ for C. glabrata HM-1123. The results further indicate an inverse correlation between the amount of Cu²⁺ additive and MFC value, suggesting that Cu²⁺ complexes with DSF and DDTC in situ to decelerate Candida growth in a fungistatic manner.

Figure 2.

Effects of copper sulfate on DSF and DDTC growth inhibition of C. glabrata HM-1123. Comparison of altered (a) growth by turbidity measurement over time and (b) fungicidal activity following 24-h treatment.

These findings led us to compare the effect of DSF and DSF + Cu²⁺ treatments by time-kill kinetics (Table 3). Comprehensive assessment with C. glabrata HM-1123 again confirmed an MFC of 8 μg ml⁻¹ for DSF (i.e., 2× MIC). Comparisons between the 24-h treatment cultures revealed that 10 μM Cu²⁺ impaired the fungicidal activity of DSF at 1× and 2× MIC, but not 4× MIC. Increased colony counts were also detected for Cu²⁺-treated cultures of C. albicans and C. auris.

Table 3.

Effect of disulfiram (DSF) and copper sulfate treatment on colony formation.

				CFU/ml (mean)
Species	Strain	DSFμg ml−1	CuSO410 μM	0 h	8 h	24 h
C. glabrata	HM-1123	0	–	2.0 × 103	6.0 × 105	1.5 × 109
		0	+	3.0 × 103	5.0 × 105	1.4 × 109
		4	–	4.0 × 103	3.0 × 103	1.2 × 103
		4	+	5.0 × 103	1.2 × 103	5.0 × 103
		8	–	3.0 × 103	2.2 × 103	0
		8	+	3.0 × 103	2.0 × 103	1.0 × 103
		16	–	4.5 × 103	1.7 × 103	0
		16	+	4.5 × 103	1.2 × 103	0
C. auris	AR0388	8	–	1.0 × 103	1.2 × 103	1.0 × 104
		8	+	2.0 × 103	2.1 × 103	1.1 × 106
C. albicans	AR0761	8	–	1.2 × 103	4.0 × 102	0
		8	+	2.5 × 103	6.0 × 102	1.0 × 102

Flow cytometry studies

Flow cytometry was employed to further characterize the effect of Cu²⁺ on the fungicidal activity of DSF and DDTC. A two stain technique with propidium iodide (PI) and SYTO 9 was used to differentiate nonviable from viable cells on the basis of membrane integrity.¹⁴ Reference plots depicted in Figure 3 (top) show nonviable C. glabrata HM-1123 cells treated with a fungicidal detergent uptake both DNA intercalating dyes (right), while viable cells with intact membranes stained single positive for SYTO 9 only (left). Comparison between the plots of reference and DSF-treated cultures revealed that the addition of Cu²⁺ to DSF resulted in a significant reduction in the nonviable PI + yeast population (middle). A similar decrease of the PI + C. glabrata population was observed when Cu²⁺ was added to the DDTC treated culture (bottom).

Figure 3.

Effect of copper sulfate on DSF (middle) and DDTC (bottom) on the viability of C. glabrata HM-1123 by flow cytometric analysis. Plots of untreated (null) and fungicidal detergent (DET) cultures were used as reference in the experiment.

Discussion

This investigation established that the addition of Cu²⁺ decreased the MICs of DSF against nine Candida species including FLU-resistant C. albicans, C. glabrata and C. auris. The data suggests that DSF + Cu²⁺ possesses synergistic potential and when the fractional inhibitory concentration indices (FICIs) are calculated for combinations up to 50 μM CuSO₄, the ΣFICI values are below 0.5 as an indicator of synergy.¹⁵ Similar results were obtained for DDTC, the initial product of DSF metabolism generated from gastric acids and thiols (e.g., glutathione) in the body. Although up to an eight-fold increase in potency was observed by differential MIC analyses, the addition of Cu²⁺ significantly antagonized the killing-effects of both agents.

These collective findings suggest that the fungicidal action of DSF is partly due to thiol-mediated generation of DDTC as a cytotoxic chelator of cellular metals. It is also believed that DSF evokes perturbations in thiol-redox homeostasis by inducing ROS-dependent cellular death.^16,17 When combined with Cu²⁺, it is plausible that generation of the hydrophobic Cu[DDTC]₂ complex facilitates the cellular uptake and accumulation of copper resulting in fungistatic effects on growth.¹¹ Future studies will therefore examine the influence of DSF and DSF + Cu²⁺ on the cellular redox state of C. albicans in an effort to define the fungicidal and fungistatic actions of the respective treatments.

Declaration of interest

The authors declare no conflicts of interest. The authors alone are responsible for the contents and writing of the paper.