Oxadiazole-Containing Macrocyclic Peptides Potentiate Azole Activity against Pathogenic Candida Species

Abstract

Opportunistic pathogens of the genus Candida reign as the leading cause of mycotic disease and are associated with mortality rates greater than 40%, even with antifungal intervention. This is in part due to the limited arsenal of antifungals available to treat systemic fungal infections. Azoles have been the most widely deployed class of antifungal drug for decades and function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. However, their utility is compromised by their fungistatic nature, which favors the development of resistance. Combination therapy has the potential to confer enhanced efficacy as well as mitigate the evolution of resistance. Previously, we described the generation of structurally diverse macrocyclic peptides with a 1,3,4-oxadiazole and an endocyclic amine grafted within the peptide backbone. Importantly, this noncanonical backbone displayed high membrane permeability, an important attribute for compounds that need to permeate across the fungal cell wall and membrane in order to reach their intracellular target. Here, we explored the bioactivity of this novel chemical scaffold on its own and in combination with the azole fluconazole. Although few of the oxadiazole-containing macrocyclic peptides displayed activity against Candida albicans on their own, many increased the efficacy of fluconazole, resulting in a synergistic combination that was independent of efflux inhibition. Interestingly, these molecules also enhanced azole activity against several non-albicans Candida species, including the azole-resistant pathogens Candida glabrata and Candida auris This work characterizes a novel chemical scaffold that possesses azole-potentiating activity against clinically important Candida species.IMPORTANCE Fungal infections, such as those caused by pathogenic Candida species, pose a serious threat to human health. Treating these infections relies heavily on the use of azole antifungals; however, resistance to these drugs develops readily, demanding novel therapeutic strategies. This study characterized the antifungal activity of a series of molecules that possess unique chemical attributes and the ability to traverse cellular membranes. We observed that many of the compounds increased the activity of the azole fluconazole against Candida albicans, without blocking the action of drug efflux pumps. These molecules also increased the efficacy of azoles against other Candida species, including the emerging azole-resistant pathogen Candida auris Thus, we describe a novel chemical scaffold with broad-spectrum bioactivity against clinically important fungal pathogens.

Keywords: Candida; Candida albicans; antifungal; azole; fluconazole; fungal pathogens; macrocycle; oxadiazole.

FIG 1

Select oxadiazole-containing macrocyclic peptides increase fluconazole efficacy against C. albicans. Checkerboard analysis of antifungal activity was performed with a combination of fluconazole and oxadiazole-containing macrocyclic peptides. C. albicans (SN95, CaLC239) was exposed to the indicated 2-fold serial dilutions of each compound for 24 h at 30°C in YPD medium. Optical densities were averaged for duplicate measurements and normalized relative to the no-drug control. Growth is quantitatively displayed by color, with green representing robust growth and black representing no growth (see color legend at bottom left). Chemical structures of the indicated macrocycles are provided to the upper left of each checkerboard. Compounds that display synergistic effects are indicated by an FICI value reported on the upper right of the checkerboard determined on the basis of calculations performed as described previously (15).

FIG 2

Oxadiazole-containing macrocyclic peptides do not impede C. albicans filamentation or multidrug efflux but do possess broad-spectrum ergosterol biosynthesis inhibitor-potentiating activity. (A) The effect of JRF1199-1 and JRF1199-2 on filamentation of C. albicans (SN95, CaLC239) was monitored after incubation in YPD medium at 30°C or 39°C for 4 h with shaking. Images were taken by differential interference contrast microscopy. Representative fields from micrographs obtained at the same magnification for all images are presented. (B) C. albicans (Caf2-1, CaLC2742) was grown in YPD medium at 30°C for 3 h with or without 125 μM macrocycle or 10 μg/ml of beauvericin. A concentration of 1 μg/ml of rhodamine-6G was added to the cultures for another 30 min at 30°C. Cells were washed twice with phosphate-buffered saline (PBS), followed by fluorescence microscopy to monitor rhodamine-6G accumulation in cells. (C) The fluorescence of untreated cells or of cells treated with beauvericin or JRF1198-2 was quantitated by flow cytometry. Assays were performed using 250 μl of culture per well, and fluorescence was measured using the FL2 (phycoerythrin) channel in a CytoFLEX flow cytometer (Beckman Coulter Inc.) with at least 20,000 events acquired per sample. Events were gated to capture at least 90% of the entire population analyzed, discarding clumps/cellular debris. Histograms representative of gated events are shown. (D) Fluconazole dose-response assays for diverse Candida species were conducted in YPD medium without (−) or with the indicated macrocycle (62.5 μM). Growth was measured by absorbance at 600 nm after 24 h (C. parapsilosis [CpLC573], C. auris [Ci 6684, CauLC5083], and C. glabrata [BG2, CgLC1002]) or after 48 h (C. tropicalis [CtLC573]) at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no-drug control well for each strain. Growth is quantitatively displayed by color, with green representing robust growth and black representing no growth (see color legend). (E) Dose-response assays against a wild-type strain of C. albicans (SN95, CaLC239) were performed in YPD medium at 30°C, and absorbance at 600 nm was measured after 24 h as described for Fig. 1. These assays were performed with fixed subinhibitory concentrations of cell membrane/wall stressor as follows: terbinafine 0.3125 μg/ml, amphotericin B (Amp B) 0.24 μg/ml, sodium dodecyl sulfate (SDS) 0.0078% [mass/vol], caspofungin 0.03125 μg/ml, calcofluor white (CFW) 15 μg/ml, and fluconazole 0.25 μg/ml. (F) Macrocycle cytotoxicity profiling using RAW 264.7 murine macrophages was performed at 72 h, using quadruplicate wells and a standard resazurin dye reduction cell viability assay. The novel macrocycles showed modest cytotoxicity over a broad concentration range compared to control macrocycles rapamycin and tacrolimus (FK506).