Potent Synergistic Interactions between Lopinavir and Azole Antifungal Drugs against Emerging Multidrug-Resistant Candida auris

Abstract

The limited therapeutic options and the recent emergence of multidrug-resistant Candida species present a significant challenge to human medicine and underscore the need for novel therapeutic approaches. Drug repurposing appears as a promising tool to augment the activity of current azole antifungals, especially against multidrug-resistant Candida auris In this study, we evaluated the fluconazole chemosensitization activities of 1,547 FDA-approved drugs and clinical molecules against azole-resistant C. auris This led to the discovery that lopinavir, an HIV protease inhibitor, is a potent agent capable of sensitizing C. auris to the effect of azole antifungals. At a therapeutically achievable concentration, lopinavir exhibited potent synergistic interactions with azole drugs, particularly with itraconazole against C. auris (fractional inhibitory concentration index [ΣFICI] ranged from 0.04 to 0.09). Additionally, the lopinavir/itraconazole combination enhanced the survival rate of C. auris-infected Caenorhabditis elegans by 90% and reduced the fungal burden in infected nematodes by 88.5% (P < 0.05) relative to that of the untreated control. Furthermore, lopinavir enhanced the antifungal activity of itraconazole against other medically important Candida species, including C. albicans, C. tropicalis, C. krusei, and C. parapsilosis Comparative transcriptomic profiling and mechanistic studies revealed that lopinavir was able to significantly interfere with the glucose permeation and ATP synthesis. This compromised the efflux ability of C. auris and consequently enhanced the susceptibility to azole drugs, as demonstrated by Nile red efflux assays. Altogether, these findings present lopinavir as a novel, potent, and broad-spectrum azole-chemosensitizing agent that warrants further investigation against recalcitrant Candida infections.

Keywords: ATP bioluminescence assay; Candida auris; HIV protease inhibitors; Nile red efflux assay; azole resistance; glucose transporters; glucose-induced acidification assay.

FIG 1

Identification of the primary screening hits that sensitized C. auris AR0390 to fluconazole. The Johns Hopkins clinical compound library was screened at 16 μM against the azole-resistant C. auris AR0390 in the absence of fluconazole (a). Hit compounds identified included lopinavir (LPV), aprepitant (APR), benzododecinium chloride (BZD), and benzalkonium chloride (BZK), which inhibited fungal growth by more than 80% in the presence of 32 μg/ml of fluconazole (b). Cultures treated with hit compounds were validated spectrophotometrically by measuring the absorbance at OD₄₉₀ nm (c).

FIG 2

Time-kill analysis of lopinavir at 10 μg/ml, fluconazole (FLC) at 16 μg/ml, or a combination of the two drugs. Test agents were evaluated against C. auris AR0390 over a 48-h incubation period at 35°C. DMSO served as a negative untreated control (a). Cultures of C. auris AR0390 treated with lopinavir (at 10 μg/ml), either alone or in combination with fluconazole (at 16 μg/ml), were spotted onto YPD agar plates and incubated for 24 h before being scanned (b).

FIG 3

Broad-spectrum synergistic interactions of lopinavir and itraconazole (ITC) against different Candida species. Shown are fractional inhibitory concentration index (ƩFICI) values as calculated from checkerboard assays (a). Cultures of C. albicans TWO743, C. krusei ATCC 14243, C. tropicalis ATCC 1369, and C. parapsilosis ATCC 22019 were treated with LPV (10 μg/ml) and ITC (0.25× MIC), either alone or in combination. Cultures were incubated at 35°C for 24 h and then were spotted onto YPD agar plates and incubated for 24 h before being scanned (b).

FIG 4

Effects of lopinavir, itraconazole, and a combination of the two drugs on the C. auris transcriptome. Shown is a transcriptional comparison of C. auris AR0390 treated with LPV, ITC, or a combination of both drugs versus the untreated control. (a) Heat map of FPKM values of DEGs of each treatment versus the untreated control, scaled by row. Genes were clustered using hierarchical clustering based on Euclidean distance. (b) Volcano plot of DEGs from C. auris AR0390 treated with LPV at 10 μg/ml. (c) Volcano plot of DEGs from C. auris AR0390 treated with ITC at 1 μg/ml. (d) Volcano plot of DEGs from C. auris AR0390 treated with the lopinavir/itraconazole (LPV/ITC) combination.

FIG 5

Gene Ontology (GO) enrichment analysis of differentially expressed genes in the lopinavir/itraconazole-treated sample. GO analysis was implemented by the Cluster Profiler R package, and a P value of ≤0.05 was used as the cutoff parameter. Upregulated GO terms (a) and downregulated GO terms (b) are shown.

FIG 6

Effect of itraconazole, lopinavir (LPV), or a combination of the two drugs on the mRNA expression levels of five selected genes involved in glucose transport and efflux in C. auris. Exponentially grown C. auris AR0390 cells were treated with either LPV (10 μg/ml), ITC (1 μg/ml), or a combination of the two drugs for 3 h. Following treatment, cells were harvested and lysed, and RNA was extracted. The expression of genes encoding glucose transport (HGT6 and HGT8) and the azole resistance-related efflux genes (CDR1, CDR2, and MDR1) was internally normalized to ACT1 and compared to that of the untreated control. Asterisks indicate significant changes in expression of the examined genes (at least 2-fold differences, up or down) relative to that of the untreated control. The results are presented as means ± SD.

FIG 7

Effect of lopinavir on the glucose utilization, ATP content, and efflux activity of C. auris. (a) Effect of LPV on the glucose utilization ability of C. auris. Cultures of C. auris AR0390 were treated with DMSO (1%) or lopinavir (LPV) at 10 μg/ml, and then the ability of C. auris to utilize externally supplemented glucose and acidify the assay medium was detected by the decreased absorbance of bromophenol blue at 590 nm. (b) Effect of lopinavir on the ATP content of C. auris. Exponentially grown C. auris AR0390 cells were treated with DMSO (1%) or LPV (10 μg/ml) for 3 h at 35°C before being lysed, and ATP content was determined. Asterisks represent a statistical difference (P < 0.05) in ATP content between treated cells and the untreated control, as determined by unpaired t test. (c) Effect of lopinavir (10 μg/ml) on Nile red efflux from 10 different C. auris isolates. (d) Effect of lopinavir (at 2, 8, or 32 μg/ml) on Nile red efflux from recombinant S. cerevisiae strains expressing individual efflux genes CDR1, CDR2, or MDR1 from C. albicans. Data represent means + SD from triplicate measurements. Asterisks represent a statistical difference (P < 0.05) in the efflux of Nile red for LPV-treated cells compared to that of the untreated control, as determined by multiple t tests using the Holm-Sidak method for multiple comparisons.

FIG 8

In vivo efficacy of the lopinavir/itraconazole combination using a Caenorhabditis elegans infection model. Nematodes infected with C. auris AR0390 were treated with LPV at 10 μg/ml and ITC at 1 μg/ml, either alone or in combination. Untreated worms served as a negative control. Effects of LPV, ITC, and the lopinavir/itraconazole combination on reducing fungal burden (CFU) after 24 h of treatment are shown (a). Asterisks indicate a statistical significance (P < 0.05) compared to the untreated control, while a pound sign indicates a statistical significance for the combination treatment compared to treatment with ITC alone (P value < 0.05, as determined by one-way analysis of variance [ANOVA] using Dunnett’s test for multiple comparisons). A Kaplan-Meier survival curve, assessed by log-rank test for significance, to evaluate the ability of the lopinavir/itraconazole combination to enhance survival of C. elegans infected with C. auris AR0390 was performed (b).