Programmed cell death in Naegleria fowleri induced by frutescinol acetate, a polyacetylene from Argyranthemum frutescens

BACKGROUND: Primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri remains a highly lethal infection with limited therapeutic options. PURPOSE: In the search for novel amoebicidal compounds of natural origin, this study investigates the mechanism of action of a bioactive metabolite from Argyranthemum frutescens, a Canary Islands endemic plant, against Naegleria fowleri. STUDY DESIGN: The present study combined bio-guided fractionation of A. frutenscens to isolate amoebicidal compound followed by elucidation of it mechanism of action against N. fowleri. METHODS: Fractionation of the ethanolic root extract was performed to isolate the bioactive compounds. Activity was tested against N. fowleri in both trophozoite and cyst forms (strains ATCC® 30,808™ and ATCC® 30,215™) and compared to the reference drug miltefosine. Cytotoxicity studies were conducted in a murine macrophage cell line. The onset of programmed cell death in treated cells was investigated through the analysis of keys cellular events including, chromatin condensation, plasma membrane permeability, mitochondrial function, and reactive oxygen species production. RESULTS: The bio-guided fractionation afforded the frutescinol acetate, a polyacetylene that showed strong activity against N. fowleri. Half maximal inhibitory concentration (IC₅₀) values were 4.97 µM and 5.70 µM for trophozoite strains ATCC® 30,808™ and ATCC® 30,215™, respectively, exceeding the potency of miltefosine. The compound was also active against N. fowleri cysts, with an IC₅₀ of 8.53 µM. Cytotoxicity studies revealed a favorable selectivity profile. The compound triggered a cascade of cellular events associated with programmed cell death, including chromatin condensation and increased plasma membrane permeability. These alterations led to mitochondrial dysfunction, overproduction of reactive oxygen species, and consequent oxidative stress. CONCLUSION: Frutescinol acetate exhibits potent amoebicidal activity and induces programmed cell death-like processes in N. fowleri, supporting its potential as a lead for further pharmacological evaluation.