Demethoxycurcumin exhibits amoebicidal activity against Acanthamoeba triangularis trophozoites and cysts and inhibits...

Acanthamoeba is a ubiquitous free-living protist commonly found in soil and water, with the T4 genotype responsible for most human infections. Treatment remains challenging due to the limited efficacy of current therapeutics and the parasite's ability to form a highly resistant double-walled cyst. In this study, we investigated the anti-Acanthamoeba potential of demethoxycurcumin, a bioactive curcumin derivative, using A. triangularis, a clinically relevant T4 genotype species. Among the curcumin derivatives tested, demethoxycurcumin exhibited the strongest amoebicidal activity, effectively targeting both trophozoite and cyst forms. Scanning electron microscopy revealed pronounced ultrastructural damage, including loss of acanthopodia, membrane disruption, and pore formation, indicating compromised cellular integrity. Notably, demethoxycurcumin significantly inhibited encystation under starvation conditions, maintaining the parasite in the trophozoite stage. Consistently, transcriptional analysis showed that key autophagy-related genes (AcATG3, AcATG8b, AcATG12, and AcATG16) remained close to basal levels following sublethal treatment, supporting suppression of autophagy-associated encystation. Molecular docking and dynamics simulations showed that demethoxycurcumin binds stably to Vps34 and Cdc2b of Acanthamoeba spp., forming key hydrogen bonds with LYS40, GLU90, LEU92, ALA153, and PHE155, along with π interactions that support enzymatic regulation. Compared to curcumin, demethoxycurcumin formed fewer but significant contacts, maintaining persistent binding and moderate flexibility over 100 ns, highlighting its potential as a selective modulator of autophagy and cell cycle pathways. Complementary network pharmacology analyses identified overlapping targets between demethoxycurcumin and A. triangularis infection-related proteins, highlighting hub genes such as AKT1, TNF, MMP9, CDK1, and PIK3C3, and enriched pathways in autophagy, immune regulation, oxidative stress responses, and kinase-mediated signaling. Collectively, these findings suggest that demethoxycurcumin exerts anti-Acanthamoeba activity through coordinated disruption of autophagy and cell-cycle regulatory networks, leading to arrest and impaired encystation of Acanthamoeba trophozoites.