Derivatives of the Antimalarial Drug Mefloquine Are Broad-Spectrum Antifungal Molecules with Activity against Drug-Resistant Clinical Isolates

Abstract

The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multidrug-resistant pathogens, such as Candida auris Here, we report that derivatives of the antimalarial drug mefloquine have broad-spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of antifungal drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles obtained using human cell lines indicated that the toxicity profiles of the mefloquine derivatives are very similar to those of the parent mefloquine, despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, subinhibitory concentrations of the mefloquine derivatives inhibited the expression of virulence traits, including filamentation in Candida albicans and capsule formation/melanization in Cryptococcus neoformans Mode/mechanism-of-action experiments indicated that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multitarget mechanism of action. The broad-spectrum scope of activity, blood-brain barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

Keywords: Aspergillus; Candida; Candida auris; Cryptococcus; Cryptococcus neoformans; antifungal; antifungal agents; mefloquine; repurposing.

FIG 1

Chemical structures of mefloquine (A) and mefloquine derivatives obtained from the National Cancer Institute Chemical Repository (B).

FIG 2

The combination of mefloquine and mefloquine derivatives with fluconazole is fungicidal against Cryptococcus neoformans. (A) The combination of fungistatic concentrations of mefloquine (at one-quarter the MIC) and fluconazole (at one-half the MIC) is fungicidal for C. neoformans strain H99 incubated in YPD at 37°C. (B) All of the mefloquine derivatives except for 13480 showed similar fungicidal activity in combination with fluconazole under the conditions described above. Samples of each culture were plated on YPD at the indicated time points, and the numbers of CFU per milliliter were quantified using serial dilutions after incubation at 30°C. The curves represent the results of single experiments representative of those of at least two independent experiments. Error bars indicate the standard deviations from two technical replicates.

FIG 3

The in vitro toxicities of mefloquine and mefloquine derivatives against two cell lines are similar. The indicated cell lines, HepG2 (A) and A549 (B), were treated with DMSO or the indicated mefloquine derivative. The metabolic activity of the cells was determined by measuring the reduction of a metabolically active dye (XTT) and compared to that of the untreated controls over the indicated range of drug concentrations. The curves are representative of those from three independent experiments, and the error bars indicate the standard deviations from three technical replicates. The table below panels A and B lists the half-maximal (50%) inhibitory concentrations (IC₅₀) against the indicated human cell lines.

FIG 4

Mefloquine and mefloquine derivatives inhibit C. albicans filamentation at sub-growth-inhibitory concentrations. Candida albicans strain SC5314 was inoculated into YPD containing drug or DMSO and incubated for 1 h at 37°C with shaking. The cultures were supplemented with 1% fetal bovine serum and grown at 37°C with shaking for 1 h to induce filamentation. Samples of the cultures were stained with propidium iodide, harvested, and examined by microscopy. (A) Bright-field images of live C. albicans cells show that mefloquine and the mefloquine derivatives prevent filamentation. (B) Quantitative analysis was performed by visually inspecting the microscopy images of live cells in the yeast, pseudohyphal, and hyphal forms. (C) Quantitative analysis of live C. albicans cells indicates that filamentation is affected by mefloquine in a dose-dependent manner.

FIG 5

Mefloquine and mefloquine derivatives suppress the expression of C. neoformans virulence traits. (A and B) Cryptococcus neoformans strain H99 was incubated in DMEM at 37°C in 5% CO₂ in the presence of DMSO or the indicated concentration of the mefloquine derivative (at one-half to one-quarter the MIC). The cells were harvested and stained with India ink. The cells were photographed (A), and the number of cells with or without a capsule was counted (n > 100 cells per experiment) and converted into a percentage. The bars in panel B indicate the means from two or three independent experiments, and the error bars are the standard deviations. (C) A series of 10-fold dilutions of H99 was spotted onto plates containing l-DOPA medium as well as the indicated concentration of mefloquine or the mefloquine derivative, incubated at 37°C for 48 h, and photographed. The photograph is representative of the photographs from two independent replicates.

FIG 6

Mefloquine derivatives disrupt vacuolar morphology. Exponential-phase cultures of S. cerevisiae reference strain BY4741 were exposed to DMSO or the indicated drug concentrations in YPD at 30°C for 2 h. Concurrently, a culture of S. cerevisiae with a vps1Δ mutation (BY4741 background) was also grown in YPD at 30°C for 2 h without drug or DMSO. All cells were harvested and stained with FM4-64 to visualize the vacuole and photographed. The vacuolar morphology of the treated cultures was similar to the pattern reported for the vps1Δ strain. Propidium iodide staining of the same cultures showed that >95% of cells were viable at the time of processing.

FIG 7

Mefloquine derivatives dissipate the proton motive force across the mitochondrial membrane. (A) Candida albicans strain SC5314 cells were treated with the indicated concentration of MEF derivative 13480 or DMSO for 2 h in YPD at 37°C. The cells were harvested, stained with either MitoTracker Red CMXRos or MitoTracker Green, and imaged. Identical exposure settings were used for both samples. Propidium iodide staining indicated that >95% of the cells were viable at the time of imaging. (B) Cryptococcus neoformans strain H99 cells were processed as described in the legend to panel A but for MitoTracker Red staining only. (C) Treatment with the other mefloquine derivatives showed a decreased uptake of MitoTracker Red by Candida albicans strain SC5314.

FIG 8

Mefloquine derivatives induce mitochondrial DNA instability in C. glabrata. (A) Candida glabrata strain KK2001 was incubated with MEF derivative 305758 overnight in YPD medium in an MIC plate with a 2-fold sequential dilution format. The MIC was read at 24 h (8 μg/ml). After 48 h, the surviving cells were plated on YPD, incubated at 30°C, and photographed. The black arrows indicate small or petite mutant colonies, while the white arrows indicate normal-sized colonies. (B) Small and normal-sized colonies were isolated under the conditions described above, patched on YP–2% glycerol (YPG) medium, and incubated at 30°C for 2 days and then imaged.

FIG 9

C. glabrata petite mutant formation modulates the activity of mefloquine derivative-fluconazole combinations. (A) Checkerboard assays with Candida glabrata strain BG2 were performed with MEF derivative 305758 and fluconazole in YPD and YPG media at 37°C. After 24 h of incubation, the optical density of each well was measured and compared to that of the DMSO-only well. The percentage of growth inhibition was calculated and is shown both numerically and in heat map form. (B) The surviving cells from a well with approximately 50% growth inhibition (32 μg/ml FLC plus 1 μg/ml 305758) and cells from the DMSO-only well were plated on YPD and incubated for 2 days at 30°C. Drug-treated cells produced a majority of petite mutant colonies, with few normal-sized colonies being present. (C) A Candida glabrata rho-positive (rho⁺) strain KK2001 and a rho-negative (rho⁻) derivative were stained with DAPI. The rho⁺ strains show a diffuse staining pattern indicative of both mitochondrial and nuclear DNA staining (indicated by the large arrowhead), while the rho⁻ strains show a focal nuclear pattern indicative of nuclear staining only (indicated by the thin arrow). From the FIC well containing 32 μg/ml FLC plus 1 μg/ml 305758, normal-sized colony cells from panel B stain in a pattern consistent with rho⁺, while the petite mutant colony cells from panel B show the focal pattern consistent with rho⁻.