Mechanism of Methylene Blue Inducing the Disulfide Bond Formation of Tubulin-Associated Unit Proteins.

Methylene blue (MB) has recently completed a Phase-3 clinical trial as leuco-methylthioninium (LMT) bis(hydromethanesulfonate) for treating Alzheimer's disease. Herein, we investigated the mechanism underlying the MB inhibition of tubulin-associated unit (tau) aggregation by focusing on tau monomers. We found that MB causes disulfide bond formation, resulting in strong nuclear magnetic resonance chemical shift perturbations in a large area of tau proteins. The oxidized form of MB, namely methylthioninium (MT), specifically catalyzed the oxidation of cysteine residues in tau proteins to form disulfide bonds directly using O. This process is independent of the MT-to-LMT redox cycle. Moreover, MT preferentially oxidized C291 and C322 in the lysine-rich R2 and R3 domains. Under brain physoxia conditions, LMT may convert to MT, possibly interfering with tau fibrillation via disulfide bond formation.