Identification of Antifungal Compounds against Multidrug-Resistant Candida auris Utilizing a High-Throughput Drug-Repurposing Screen

Abstract

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

Keywords: Candida auris; drug combination therapy; drug repurposing; multidrug resistance.

FIG 1

Workflow and optimization. (A) Scheme of anti-Candida auris susceptibility assay. Candida auris harvested from colonies grown on Sabouraud dextrose agar (SDA) were incubated in RPMI medium with compounds to identify inhibitors. (B and C) Optimization of the ATP-based antifungal susceptibility assay by fungal density and incubation time. Cells were diluted with fresh medium into ratios of 1 to 1,000, 1 to 500, and 1 to 200 prior to dispensing into assay plates. (B) Fungal growth was detected by an ATP-based assay at 24 and 48 h. (C) Two known compounds, posaconazole and micafungin, were tested against strains 0384 and 0390 to serve as a positive control.

FIG 2

Concentration-response curves. (A) Concentration-response curves of compounds with pan-activity for all tested strains. Each data point is presented as mean ± standard deviation (SD) for three independent experiments. (B) Concentration-response curves of myriocin with or without flucytosine. Each data point is presented as mean ± standard deviation (SD) for two experiments.

FIG 3

Schematic representation of the sphingolipid pathways in fungi. All of these reactions are reversible. A dotted line means additional enzymatic steps are present but are not illustrated for clarity. DHS, dihydrosphingosine; PHS, phytosphingosine; DHS-1-P, dihydrosphingosine-1-phosphate; PHS-1-P, phytosphingosine-1-phosphate; DHC, dihydroceramide; PHC, phytoceramide; CER, ceramide; OH-PHC, hydroxylated-phytoceramide; OH-CER, hydroxylated ceramide; GlcCer, glucosylceramide; IPC 36, inositol phosphoryl ceramide containing 36 carbons (sphingoid base plus fatty acid); IPC 42, inositol phosphoryl ceramide containing 42 carbons (sphingoid base plus fatty acid); IPC-44, inositol phosphoryl ceramide containing 44 carbons (sphingoid base plus fatty acid).