Identification of a Phenylthiazole Small Molecule with Dual Antifungal and Antibiofilm Activity Against Candida albicans and Candida auris

Abstract

Candida species are a leading source of healthcare infections globally. The limited number of antifungal drugs combined with the isolation of Candida species, namely C. albicans and C. auris, exhibiting resistance to current antifungals necessitates the development of new therapeutics. The present study tested 85 synthetic phenylthiazole small molecules for antifungal activity against drug-resistant C. albicans. Compound 1 emerged as the most potent molecule, inhibiting growth of C. albicans and C. auris strains at concentrations ranging from 0.25-2 µg/mL. Additionally, compound 1 inhibited growth of other clinically-relevant yeast (Cryptococcus) and molds (Aspergillus) at a concentration as low as 0.50 µg/mL. Compound 1 exhibited rapid fungicidal activity, reducing the burden of C. albicans and C. auris below the limit of detection within 30 minutes. Compound 1 exhibited potent antibiofilm activity, similar to amphotericin B, reducing the metabolic activity of adherent C. albicans and C. auris biofilms by more than 66% and 50%, respectively. Furthermore, compound 1 prolonged survival of Caenorhabditis elegans infected with strains of C. albicans and C. auris, relative to the untreated control. The present study highlights phenylthiazole small molecules, such as compound 1, warrant further investigation as novel antifungal agents for drug-resistant Candida infections.

Figure 1

Time-kill analysis of compound 1, itraconazole, and amphotericin B. Test agents were evaluated against (a) C. albicans P60002 and (b) Candida auris strain 390 over a 24-hour incubation period at 35 °C. DMSO served as a negative control. Error bars represent standard deviation values. Dashed horizontal lines (---) represents the limit of detection (LOD) for the assay.

Figure 2

Inhibition of Candida biofilm formation. Compound 1 and amphotericin B were evaluated for their ability to inhibit biofilm formation by (a) C. albicans P60002 and (b) C. auris strain 390. An overnight suspension of C. albicans was diluted in RPMI-1640 medium supplemented with MOPS (to achieve a starting inoculum ~5 × 10⁵ CFU/mL) and exposed to compound 1 or amphotericin B (at the indicated concentrations) or left untreated for 24 hours at 35 °C to permit biofilm formation. The biofilm was stained with 0.1% crystal violet, de-stained with ethanol, and biofilm mass quantified at OD₅₉₅. Data represents percent inhibition of biofilm mass by compound 1 relative to the untreated control. Data were analyzed via an unpaired one-way t-test (P < 0.05). Asterisks (*) indicate statistical difference between amphotericin B-treated and compound 1-treated wells.

Figure 3

Antibiofilm activity of compound 1 and amphotericin B against mature Candida biofilm. Test agents were evaluated against (a) C. albicans P60002 and (b) C. auris strain 390 adherent biofilm evaluated with the XTT assay. Adherent biofilms were treated with either compound 1 or amphotericin B (both in triplicate) at the concentrations presented, over a 24-hour period. The percent metabolic activity for each treatment was calculated relative to untreated wells. Error bars represent standard deviation values. Asterisk denotes statistical difference between compound 1 and amphotericin B relative to the negative control (untreated wells) evaluated using a two-way ANOVA, with post hoc Dunnet’s test for multiple comparisons (P < 0.05).

Figure 4

Toxicity of compound 1 against monkey kidney epithelial cells (Vero). Cells were exposed to compound 1 for 24 hours. Data represent percent viable cells after exposure to the compound (tested in triplicate) at 8, 16, 32, and 64 μg/mL using the MTS assay. Dimethyl sulfoxide (DMSO) was used as a negative control. Error bars represent standard deviation values. An asterisk (*) denotes statistical difference between compound 1 and DMSO evaluated using a two-way ANOVA, with post hoc Sidak’s multiple comparisons test (P < 0.05).

Figure 5

Toxicity of compound 1 and efficacy of compound 1 and 5-Fluorocytosine in C. elegans infected with C. albicans or C. auris. (a) Adult (L4-stage) worms were exposed to compound 1 at four different concentrations and viability was recorded daily for four days. Adult (L4-stage) worms were infected with the highly-virulent, fluconazole-resistant strains (b) C. albicans P60002 or (c) C. auris strain 390 for 90 minutes at 25 °C. Worms were washed and then treated with compound 1 (either at 5 µg/mL or 10 µg/mL), 5-Fluorocytosine (5-FC, 5 µg/mL) or left untreated. Survival of worms was monitored daily and recorded. Data are presented as a Kaplan-Meier survival curve.