Synergistic interaction of amphotericin B and betulinic acid against clinically important fungi: evidence from in vitro and in silico techniques.

Betulinic acid (BA), in combined application with amphotericin B, shows a synergistic effect against Candida, Aspergillus, Scedosporium, Fusarium, and Mucorales fungi at a concentration as low as 0.125 µg/mL. Amphotericin B showed slightly higher affinity towards BA than toward ergosterol, according to our in silico molecular docking results, explaining the observed Eagle effect. Moreover, it can bind both molecules simultaneously, suggesting the possibility of the formation of mixed pores, thus increasing the membrane-disrupting activity.IMPORTANCEThe rising incidence of invasive fungal infections, coupled with the emergence of antifungal resistance, presents a significant challenge in clinical settings. The inherent resistance of certain fungi to conventional antifungal agents, alongside the limitations posed by side effects and drug interactions, necessitates the exploration of alternative therapeutic strategies. This study highlights the potential of combining amphotericin B (AmB) with betulinic acid (BA) to enhance antifungal efficacy against clinically relevant pathogens, including Candida albicans and Aspergillus fumigatus, as well as mucormycosis-causing fungi. The results demonstrate the synergistic interactions between AmB and BA, which effectively inhibited fungal growth at lower concentrations and are within reported serum levels. In silico molecular docking studies further support the hypothesis that BA may facilitate AmB's mechanism of action, potentially leading to increased pore formation in fungal membranes.