Repurposing Acetylcholinesterase Inhibitors for Leishmaniasis: Donepezil Hydrochloride and Related Compounds Against the American Tegumentary Form

<b>Background/Objective:</b> American tegumentary leishmaniasis is a neglected tropical disease with limited therapeutic options characterized by high toxicity and poor tolerability. Drug repurpose offers a pragmatic strategy to accelerate the development of safer treatments. This study evaluated the antileishmanial activity of three clinically approved acetylcholinesterase (AChE) inhibitors-donepezil hydrochloride (DH), rivastigmine tartrate (RT), and galantamine hydrobromide (GH), tested individually and in combination with amphotericin B (AmpB) against <i>Leishmania</i> species relevant to tegumentary leishmaniasis. <b>Methods:</b> Antileishmanial activity was assessed against <i>Leishmania (Leishmania) amazonensis</i> promastigotes and intracellular amastigotes and <i>Leishmania (Viannia) braziliensis</i> promastigotes and axenic amastigotes. Cytotoxicity was evaluated in mammalian cell lines. The synergy with AmpB was analyzed at different proportions. Mechanistic studies included morphological analysis using light and scanning electron microscopy, flow cytometry, AChE activity assays, choline supplementation experiments, and membrane fluidity measurements. <b>Results:</b> All three AChE inhibitors demonstrated antileishmanial activity with selectivity indices > 1. DH emerged as the most promising candidate (IC₅₀ = 16.82 μM against promastigotes; SI = 10.25), with superior potency compared to other repurposed drugs. Strong synergistic interactions with AmpB were observed for all inhibitors (χΣFIC ≤ 0.17), with DH-AmpB displaying the most robust synergy (χΣFIC = 0.09), reducing the IC ₅₀ of AmpB by nearly 90-fold. DH induced distinct morphological alterations and acted through non-cholinergic mechanisms. The DH-AmpB combination retained maximal efficacy against <i>L. (V.) braziliensis</i>, with enhanced activity against clinically relevant amastigotes. <b>Conclusions:</b> Repurposed AChE inhibitors, particularly donepezil hydrochloride, are highly promising therapeutic candidates for tegumentary leishmaniasis. The robust synergistic effect with amphotericin B, together with their favorable safety profiles and non-antimicrobial mechanisms, positions these drugs as viable partners in dose-sparing combination regimens that could improve treatment adherence and reduce toxicity in endemic areas.