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 O₂. 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 in vivo brain physoxia conditions, LMT may convert to MT⁺, possibly interfering with tau fibrillation via disulfide bond formation.