Harnessing Inhibition of Efflux to Reverse Antifungal Resistance

Video Transcript

Antifungal resistance, commonly via efflux overexpression, poses an enormous risk to global health. Many pathogens, including Candida albicans, have evolved to overexpress efflux pumps to allow fungal cells to expel antifungals. The goal of my project was to repurpose existing compounds to reverse antifungal resistance by inhibiting efflux.

I found two compounds, doramectin and URMC-099-C, that restore the activity of the antifungal fluconazole against a fluconazole-resistant strain of Candida albicans via efflux inhibition.

I found that doramectin has minimal cytotoxicity against the standard human liver cell line HepG2 at effective antifungal potentiator concentrations.

I also discovered that both of the compounds enhance the inhibition of Candida albicans filamentation by fluconazole.

Therefore, doramectin is a highly promising compound that can be repurposed as a novel efflux inhibitor to reverse antifungal resistance.

Why?

Problem

Fungal pathogens lead to almost 4 million deaths per year, and many of the world's most dangerous pathogens are developing antifungal resistance, further exacerbating this issue. The main mechanism of antifungal resistance is overexpression of efflux pumps, allowing fungal cells to expel antifungals. Currently, there are no efflux inhibitors approved for clinical use as antifungals in humans.

Solution

Repurposing existing compounds tends to result in much lower development costs (averaging $300 million for repurposed compounds versus $2 billion for novel drug development) and the introduction of the compounds to market in a shorter timespan (3-12 years versus 10-17). Thus, this project aimed to repurpose existing compounds to restore the activity of the commonly used antifungal fluconazole against a fluconazole-resistant strain of Candida albicans, inhibiting efflux overexpression and reversing antifungal resistance.

Why Candida albicans and fluconazole?

Candida albicans is a potentially lethal fungal pathogen that commonly overexpresses efflux pumps to expel antifungals. It is one of the most common pathogens infecting humans. Fluconazole is the most widely used antifungal for Candida infections and its activity is completely disabled by efflux overexpression as it requires intracellular accumulation.

Hypotheses

Few of the tested compounds would have no single agent activity but synergize with fluconazole.

Even fewer of the previous compounds would be efflux inhibitors.

The efflux inhibitors would not be cytotoxic to human liver cells, targeting a nonessential process in human cells.

Because the efflux system is complex, it may be difficult for the compounds to penetrate filamenting fungi.

Methods

Medicines for Malaria Venture (MMV) Compound Screen

640 compounds from the MMV Pandemic Response and Global Health Priority boxes were screened.

Compounds that did not have single agent antifungal activity but that synergized with fluconazole (FLC) against an FLC-resistant strain of Candida albicans (CaCi-17) were identified to uncover potential efflux inhibitors. An efflux inhibitor would not kill fungal cells by itself but would restore the activity of other antifungals.

Nile Red Accumulation Assay

Efflux-overexpressing CaCi-17 cells were treated with titrations of compounds (50µM to 3.125µM) and compared to no compound, non-inhibitor dimethyl sulfoxide (DMSO), and efflux inhibitor azoffluxin controls.

If the compounds were efflux inhibitors, the intracellular accumulation of the Nile Red dye would be greater and the cells would fluoresce.

Cell fluorescence averages for each compound concentration were analyzed qualitatively and quantitatively.

Cytotoxicity Assay

The liver metabolizes drugs, so compounds for clinical use should have minimal cytotoxicity to human liver cells.

The cell viability of the standard human liver cell line HepG2 following titration treatments starting with 50µM of compound was analyzed.

Viability was compared to the non-toxic DMSO solvent control and the toxic topotecan control.

Cytotoxicity was analyzed via an alamarBlue metabolic readout.

Filamentation Assay

Filamentation is a critical virulence factor in Candida albicans, and the cells were triggered to filament by cues including increased heat and the presence of serum.

Evaluated the efflux inhibitors' ability to inhibit filamentation in CaCi-17 by themselves.

Evaluated the compounds' ability to enhance the filamentation inhibition in CaCi-17 by fluconazole when used in combination with the antifungal.

Results

Medicines for Malaria Venture Screen

The heat maps in the first image (see images above) show the 11 most growth-inhibitory compounds identified by optical density quantification compared to the solvent control DMSO and the efflux inhibitor control azoffluxin. The compounds did not have antifungal activity by themselves but synergized with FLC.

Testing hit compounds in a dose-response assay confirmed that doramectin, URMC-099-C, and PF-3450074 potentiate FLC activity against FLC-resistant CaCi-17. The double agent antifungal activity of PF-3450074 is limited at concentrations lower than 25µM, and URMC-099-C has activity at concentrations above 3.125µM. Doramectin maintains promising double agent antifungal activity at concentrations of 1.5625µM and above. Interestingly, the activity of doramectin + FLC is higher at 25µM than 50µM, suggesting higher concentrations may result in a reactionary stress and thus defense response in the cell.

This assay revealed potential efflux inhibitors that were then tested to identify compounds that inhibit efflux in the Nile Red accumulation assay.

Nile Red Accumulation Assay

In the second image, the orange fluorescing cells indicate the efflux activity is inhibited, thus the cells are unable to pump out the Nile Red dye.

Compared to the non-inhibitor control DMSO and the control inhibitor azoffluxin, doramectin and URMC-099-C are efflux inhibitors, as identified by the orange fluorescing cells, providing a strategy to overcome drug resistance.

In third image, the graphs quantify cell fluorescence averaged across hundreds of cell counts for each of the tested compound concentrations. At their highest tested concentrations (50µM), URMC-099-C inhibits efflux on par with azoffluxin, and doramectin approximately tenfold more, demonstrating that doramectin is the most potent efflux inhibitor. Even at the lowest tested concentration of 3.125µM, doramectin still inhibits efflux just below the level of 25µM of azoffluxin.

Cytotoxicity Assay

As visible in the fourth image, URMC-099-C is relatively cytotoxic across all concentrations, while PF-3450074 has low cytotoxicity to HepG2 liver cells with greater than 50% cell viability observed at the highest tested concentration. Doramectin has minimal cytotoxicity at concentrations below 4µM. This suggests doramectin is the most promising efflux inhibitor.

As novel candidates for clinical efflux inhibition of fungal pathogens in humans, it is crucial that these compounds maintain minimal cytotoxicity against human cells. This assay revealed that doramectin is both the most potent of the two efflux inhibitors and has much lower cytotoxicity than URMC-099-C.

Filamentation Assay

In the fifth image, doramectin and URMC-099-C do not inhibit filamentation on their own, as filamentous Candida albicans is visible. Doramectin and URMC-099-C enhance the ability of fluconazole to inhibit filamentation in CaCi-17 (256 µg/mL FLC + 50 µM compound), overcoming a critical virulence factor in the fungus and lessening a main mechanism of deeper tissue infection.

Discussion and Interpretation

It was found that three of the tested compounds, doramectin, URMC-099-C, and PF-3450074 enhance the activity of the antifungal fluconazole against a fluconazole-resistant strain of Candida albicans with hyperactive efflux. Doramectin and URMC-099-C show dose dependent inhibition of efflux, reversing antifungal resistance and restoring antifungal activity. Doramectin is the most inhibitory of the three compounds, demonstrating efflux inhibition approximately tenfold greater than the control efflux inhibitor azoffluxin. Compared to the toxic control topotecan, doramectin and PF-3450074 are minimally cytotoxic at lower concentrations while URMC-099-C is quite toxic across all concentrations. Doramectin and URMC-099-C enhance the ability of fluconazole to block C. albicans filamentation, overcoming a crucial virulence factor.

Conclusions

The goal of this project was achieved! Doramectin is the most promising of the three compounds studied, as it is the most potent antifungal potentiator with lower cytotoxicity. I identified a compound (doramectin) that can be repurposed as a novel efflux inhibitor to combat drug-resistant Candida albicans. The results of this project are extremely promising and can be used to reverse antifungal resistance and combat drug-resistant fungal pathogens.

Next Steps

To continue this project further, I plan to identify the specific efflux transporters the compounds are targeting in Candida albicans. I will analyze the compounds' efflux inhibition in other fungal pathogens overexpressing efflux pumps, including Candida glabrata and Cryptococcus neoformans to investigate broader inhibition. I will also analyze the effectiveness of doramectin in vivo in Caenorhabditis elegans infection models to assess activity and therapeutic potential in vivo.

Thanks

I would like to thank my mentor, Olivia Paulin, for being a source of feedback and support throughout this project, and for assisting in generating certain figures. I would also like to acknowledge Rachel Lee and Nicole Robbins for supervising my work. Thank you to the Medicines for Malaria Venture for allowing me to work with their compounds, the University of Toronto for permitting me to use their facilities, and my biosafety team for ensuring my project abides by all biosafety regulations.

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