Thiamet G is a potent, selective O-GlcNAcase inhibitor with Kiof 21 nM, while exhibiting 37, 000-fold selectivity over human lysosomal –hexosaminidase.

O-GlcNAcase (OGA):21 nM(Ki)

In NGF-differentiated PC-12 cells, inhibition of O-GlcNAcase by Thiamet G increases the cellular levels of O-GlcNAc with EC50 of approximately 30 nM. Thiamet G (100 mM) reduces tau phosphorylation by approximately 2.1-fold, 2.7-fold, 1.2-fold and 1.3-fold for Ser396, Thr231, Ser422 and Ser262, respectively. [1] Thiamet G (12.5 nM and 25 nM) significantly enhances p38 phosphorylation by increasing O-GlcNAcylation in mesangial cells. [2] In O-GlcNAc transferase or O-GlcNAcase gain of function cells, thiamet-G restores the assembly of the spindle and partially rescues histone phosphorylation. [3]

In rats, thiamet G (50 mg/kg) administrated by i.v. crosses the blood brain barrier and then results in increase in brain O-GlcNAc levels in a dose- and time-dependent manner, and reduction of tau phosphorylation in rat brain. Thiamet G is also orally bioavailable. [1] O-GlcNAc accumulation induced by thiamet G stimulates chondrogenic differentiation in C57/bl mice by increasing the gene expression of differentiation markers, as well as the activity of MMP-2 and -9. [4]

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.

Jurkat cells are seeded at 6000 cells/well in a 96-well plate, and 12 h later, cells are treated with compounds for the indicated time. Cell viability is determined by XTT assay.