Discovery of petroselinic acid with in vitro and in vivo antifungal activity by targeting fructose-1,6-bisphosphate aldolase

Background: The incidence of invasive fungal diseases (IFDs), represented by Candida albicans infection, is increasing year by year. However, clinically available antifungal drugs are very limited and encounter challenges such as limited efficacy, drug resistance, high toxicity, and exorbitant cost. Therefore, there is an urgent need for new antifungal drugs. Purpose: This study aims to find new antifungal compounds from plants, preferably those with good activity and low toxicity, and reveal their antifungal targets. Methods: In vitro antifungal activities of compounds were investigated using broth microdilution method, spot assay, hyphal growth assay and biofilm formation assay. Synergistic effects were assessed using broth micro- dilution checkerboard technique. In vivo antifungal activities were evaluated using Galleria mellonella and murine candidiasis models. Cytotoxicity of compounds was investigated using Cell Counting Kit-8 (CCK-8). Discovery and validation of antifungal targets of compounds were conducted by using monoallelic knockout library of C. albicans, , haploinsufficiency profiling (HIP), thermal shift assay (TSA), enzyme inhibitory effect assay, molecular docking, and in vitro and in vivo antifungal studies. Results: 814 plant products were screened, among which petroselinic acid (PeAc) was found as an antifungal molecule. As a rare fatty acid isolated from coriander ( Coriandrum sativum), ), carrot ( Daucus carota) ) and other plants of the Apiaceae family, PeAc had not previously been found to have antifungal effects. In this study, PeAc was revealed to inhibit the growth of various pathogenic fungi, exhibited synergistic effects with fluconazole (FLC), inhibited the formation of C. albicans hyphae and biofilms, and showed antifungal effects in vivo. PeAc was less toxic to mammalian cells. Fructose-1,6-bisphosphate aldolase (Fba1p) was identified as a target of PeAc by using HIP, TSA, enzyme inhibitory effect assay and molecular docking methods. PeAc exerted antifungal effects more effectively on fba1 Delta/FBA1 Delta /FBA1 than wild-type (WT) strain both in vitro and in vivo. . Conclusions: PeAc is an effective and low toxic antifungal compound. The target of PeAc is Fba1p. Fba1p is a promising target for antifungal drug development.