Promising Antifungal Molecules against Mucormycosis Agents Identified from Pandemic Response Box®: In Vitro and In Silico Analyses

Abstract

Mucormycosis is considered concerning invasive fungal infections due to its high mortality rates, difficult diagnosis and limited treatment approaches. Mucorales species are highly resistant to many antifungal agents and the search for alternatives is an urgent need. In the present study, a library with 400 compounds called the Pandemic Response Box^® was used and four compounds were identified: alexidine and three non-commercial molecules. These compounds showed anti-biofilm activity, as well as alterations in fungal morphology and cell wall and plasma membrane structure. They also induced oxidative stress and mitochondrial membrane depolarization. In silico analysis revealed promising pharmacological parameters. These results suggest that these four compounds are potent candidates to be considered in future studies for the development of new approaches to treat mucormycosis.

Keywords: Pandemic Response Box®; Rhizopus; antifungal agents; biofilm; in silico analyses.

Figure 1

Screening of the Pandemic Response Box^® library. A total of 400 compounds were tested against R. oryzae UCP1295. Fungal growth was quantified after incubation for 48 h via optical density and those presenting at least 60% of inhibition (dotted line) were selected.

Figure 2

Molecular structures of the effective compounds against R. oryzae UCP1295. The compounds present in the Pandemic Response Box^® library are MMV396785 (Alexidine), MMV1580844, MMV642550 and MMV019724.

Figure 3

Effect of alexidine, MMV1580844, MMV642550 and MMV019724 on biofilm formation of Rhizopus species. Fungal cells were grown on polystyrene surface in the presence of different concentrations of selected compounds (¼–8× MIC). Fungal biomass (A,D,G), extracellular matrix (B,E,H) and viability (C,F,I) were measured using violet crystal, safranin and XTT reduction assay, respectively. * p < 0.05, compared to zero (absence of drug) for each species.

Figure 4

Effect of alexidine, MMV1580844, MMV642550 and MMV019724 on preformed biofilms of three Rhizopus species. Fungal biofilm was firstly formed in RPMI 1640 medium on polystyrene surface for 24 h and then it was treated with different concentrations of selected compounds (¼–8× MIC) for another 24 h incubation. Intact fungal biofilms were considered controls (CTL, 100% biofilm) and their degradation due to treatment was compared to the control. Fungal biomass (A,D,G), extracellular matrix (B,E,H) and viability (C,F,I) were measured using violet crystal, safranin and XTT reduction assay, respectively. * p < 0.05, compared to zero (absence of drug) for each species.

Figure 5

Ultrastructural alterations of R. oryzae UCP1295 after exposure to alexidine, MMV1580844, MMV642550 and MMV019724, evaluated via scanning electron microscopy. Untreated cells (control) exhibit thicker hyphae and the formation of sporangia and spores, typical of Mucorales growth, whereas samples treated with MIC₅₀ (Table 2) values of each selected compound for 48 h show alterations in fungal surface.

Figure 6

The effect of alexidine, MMV1580844, MMV642550 and MMV019724 on R. oryzae cells analyzed using fluorescent staining. Cells were grown in the presence of ½× MIC₅₀ of each selected compound for 48 h at 37 °C. Chitin content was analyzed using calcofluor white (A). Intracellular DNA quantification was analyzed using Sytox blue staining (B). Oxidative stress; ROS was measured using DCFH-DA (C). The mitochondrial membrane polarization was measured using JC-1 (D). Neutral lipids were quantified using Nile Red stain (E). Ctl (-), a negative control that represents cells in the absence of fluorescent stain. Untreated, a positive control that represents cells stained with fluorescent stain, but without drug treatment. * p < 0.05; ** p < 0.01; *** p <0.001; ns—not significant.