Profiling fungal secondary metabolites with anti-leishmanial activity: an integrated in silico and in vitro study

Numerous reports of drug resistance in Leishmania major, the causative agent of cutaneous leishmaniasis (CL), underscore the need to discover novel therapeutics. Fungal secondary metabolites (SMs) are valuable resources for drug development and can serve as antimicrobial agents, enzyme inhibitors, and lead compounds for novel medicines. A three-dimensional model of the L. major sterol 14α-demethylase (LmCYP51) enzyme was built using L. infantum CYP51 as a template. A library of 1,167 compounds was virtually screened against LmCYP51 using high-throughput docking. Eleven promising candidates were identified and further analyzed through 100-ns molecular dynamics simulations. Six high-affinity candidates were selected for experimental validation. In vitro assays, including MTT, Giemsa staining, and flow cytometry, were performed to evaluate antiparasitic efficacy. Dihydrocitrinone emerged as the most promising compound with the most negative predicted binding free energy (ΔG = -35.41 kcal/mol) and the most potent in vitro activity, with IC₅₀ values of 9.87 and 26.29 µM against promastigotes and intracellular amastigotes, respectively. The compound exhibited moderate selectivity (selectivity index = 2.8). Flow cytometric analysis revealed that dihydrocitrinone, at its IC₅₀ concentration, promotes phosphatidylserine externalization and induces apoptosis-like programmed cell death in 86% of cells. By integrating computational prioritization with experimental validation, this study identified dihydrocitrinone, a fungal polyketide, as an early antileishmanial hit against L. major that induces apoptotic-like programmed cell death. Dihydrocitrinone's promising antileishmanial potency provides a suitable scaffold for medicinal chemistry optimization and the development of novel therapeutics for leishmaniasis. This research provides the first experimental validation of dihydrocitrinone against L. major and highlights the underexplored potential of fungal SMs in antileishmanial drug discovery.