Berbamine hydrochloride as a brain penetrant galectin 3 inhibitor in a model of Huntington's disease

Galectin-3 (Gal3) acts as an intracellular sensor of lysosomal damage and a key driver of microglial activation and neuroinflammation in multiple neurodegenerative disorders. Despite its biological importance, no inhibitors have been identified that both modulate intracellular Gal3 activity and effectively cross the blood-brain barrier (BBB). Here, we established an innovative high-content imaging platform utilizing microglia expressing Gal3-GFP, allowing direct visualization of Gal3 puncta formation upon lysosomal damage as a phenotypic readout for compound screening. Through screening of approximately 24,000 small molecules, we identified berbamine hydrochloride as a potent inhibitor of Gal3 puncta formation with confirmed BBB permeability. Mechanistically, berbamine hydrochloride disrupts Gal3 oligomerization and Gal3-TREM2 interaction via a unique allosteric mechanism distinct from the canonical glycan-binding domain. It attenuated lipopolysaccharide-induced inflammation in vitro, and achieved sustained brain exposure in vivo. In the R6/2 mouse model of Huntington's disease (HD), berbamine hydrochloride effectively improved motor functions, reduced mutant huntingtin protein aggregation, and restored crucial dopaminergic and ciliary signaling pathways. Transcriptomic profiling further identified Gal3 as a central regulatory hub within HD-associated networks that were corrected upon treatment. Computational docking and molecular dynamics simulations further supported an allosteric binding mode distinct from Gal3's canonical glycan-binding domain. Together, these findings establish berbamine hydrochloride as the first BBB-penetrant small molecule inhibitor targeting intracellular Gal3 with therapeutic benefit in HD. Furthermore, the puncta-based screening strategy we developed provides a robust platform for discovering intracellular modulators of Gal3 relevant to a wide range of neurological diseases.