In vitro activity: Thiamet G is a novel potent, and selective O-GlcNAcase inhibitor with Ki of 21 nM, it displayed 37,000-fold selectivity over human lysosomal–hexosaminidase. Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease. Thiamet G was extremely stable in aqueous solution. In nerve growth factor (NGF)-differentiated PC-12 cells, thiamet G significantly increased cellular O-GlcNAc levels with EC50 value of 30 nM in a dose dependent way.

Kinase Assay: Thiamet G is a novel potent, and selective O-GlcNAcase inhibitor with Ki of 21 nM, it displayed 37,000-fold selectivity over human lysosomal–hexosaminidase. All enzymatic assays are performed in triplicate at 37°C using 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate as substrate. 1 nM of purified OGA is incubated with the compounds for 5 min, and then 0.2 mM of the substrate is added. The liberation of 4-methylumbellifery is monitored by kinetic reading at excitation/emission 355/460 nm using a Tecan M200 plate in a mode of 60 s/cycle and 15 cycles in total.

Cell Assay: In nerve growth factor (NGF)-differentiated PC-12 cells, thiamet G significantly increased cellular O-GlcNAc levels with EC50 value of 30 nM in a dose dependent way. Thiamet G significantly reduced phosphorylation levels at Ser396 and Thr231 of Tau by 2.1-fold and 2.7-fold, respectively. Also, thiamet G decreased the phosphorylation levels at Ser422 and Ser262.