Investigating the hidden mechanism underlying the tau interaction by methylene blue

Abstract Methylene blue (MB) has recently completed another Phase-3 clinical trial as the leuco-methylthioninium (LMT) bis(hydromethanesulfonate) (LMTM) for the treatment of Alzheimer's disease (AD). We aimed to elucidate the detailed mechanisms of MB as a tau aggregation inhibitor, focusing on tau monomers and fibrils. Methylthioninium (MT + ) oxidized the Cys residues of tau proteins (2N4R, K18, and dGAE) to disulfide bonds by utilizing O 2 molecule, independent of the MB redox-cycle between MT + and LMT. The previous MB-mediated 1 H- 15 N heteronuclear single quantum coherence (HSQC) peak-broadenings in a large area (> 60 residues) of 2N4R likely resort to disulfide-centered hydrogen bonds, specific long-range interactions with the NH, and the basic side-chains in solution. In addition, at least two different MB binding sites were identified for dGAE fibrils, and the different K i values of MT + and LMT were measured against thioflavin-T (ThT), which could serve as a molecular basis to support the disaggregation activity of MB.