Synthesis of polysubstituted lactones and apoptotic hallmarks in Naegleria fowleri: Exploring their potential as...

Naegleria fowleri is the etiological agent of primary amoebic meningoencephalitis, a fulminant infection of the central nervous system that leads to death in most cases. Currently, no standardised therapy is available, and existing treatments are limited by inconsistent efficacy and significant toxicity. β-Hydroxy-γ-lactones previously described as trypanocidal and leishmanicidal agents share structural features, a reactive lactone carbonyl and a stereodefined hydroxyl group, that are commonly linked to disruption of redox and mitochondrial homeostasis in protozoan parasites. Based on this precedent, we hypothesised that the same scaffold could also be active against N. fowleri. Fourteen compounds, ten previously reported β-hydroxy-γ-lactones and four newly synthesised α, β-unsaturated-γ-lactones (butenolides, SAM-13 to SAM-16) obtained by β-elimination, were evaluated in vitro against the trophozoite and cyst stages of two N. fowleri strains (ATCC® 30808™ and ATCC® 30215™), together with cytotoxicity assays in murine macrophages (J774A.1) and human SH-SY5Y neuroblastoma cells. SAM-1 and SAM-5 emerged as the most active molecules, with low-micromolar IC₅₀ values against both trophozoites and cysts; their selectivity, however, differed markedly, with SAM-5 showing a substantially wider safety margin (selectivity index up to 12.9) than SAM-1 (selectivity index of 1.2 in neuroblastoma cells), underscoring selectivity as a key parameter for further optimisation. In contrast, three of the four newly synthesised butenolides were essentially inactive (IC₅₀ > 160 μM); the fourth, SAM-14 (derived from SAM-5), retained measurable but poorly selective activity (IC₅₀ = 56.9 μM, SI = 1.35), indicating that the requirement for the free β-hydroxyl group is substituent-dependent and providing an initial structure-activity relationship for this scaffold. Mechanistic assays showed that SAM-1 and SAM-5 induced hallmarks of programmed cell death in N. fowleri trophozoites, including phosphatidylserine externalisation, chromatin condensation, mitochondrial depolarisation, ROS overproduction, and F-actin disorganisation, together with complementary evidence of autophagic vacuole formation. In silico ADME profiling predicted favourable drug-likeness for SAM-1 and SAM-5, including compliance with Lipinski's rule of five, high gastrointestinal absorption and predicted blood-brain barrier permeability, with neither compound predicted to be a P-glycoprotein substrate. Taken together, these findings identify β-hydroxy-γ-lactones as promising, CNS-penetrant scaffolds for anti-Naegleria drug discovery and outline the structural determinants that should guide their future optimisation.