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. Here, we established an innovative high-content imaging platform using microglia expressing Gal3-GFP, allowing direct visualization of Gal3 puncta formation upon lysosomal damage as a phenotypic readout for compound screening. Through screening of ∼24 000 small molecules, we identified berbamine hydrochloride as a potent inhibitor of Gal3 puncta formation with confirmed blood-brain barrier 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, berbamine hydrochloride effectively improved motor functions, reduced mutant huntingtin protein aggregation and restored crucial dopaminergic and ciliary signalling pathways. Transcriptomic profiling further identified Gal3 as a central regulatory hub within Huntington's disease-associated networks that were corrected upon treatment. Computational docking and molecular dynamics simulations further supported an allosteric binding mode distinct from the canonical glycan-binding domain of Gal3. Together, these findings establish berbamine hydrochloride as the first blood-brain barrier-penetrant small molecule inhibitor targeting intracellular Gal3 with therapeutic benefit in Huntington's disease. Furthermore, the puncta-based screening strategy that we developed provides a robust platform for discovering intracellular modulators of Gal3 relevant to a wide range of neurological diseases.