Toluidine blue O is a potent inhibitor of human cholinesterases

Arch Biochem Biophys. 2016 Aug 15:604:57-62. doi: 10.1016/ Epub 2016 Jun 11.


In this study, the inhibitory effects of three phenothiazines [toluidine blue O (TBO), thionine (TH) and methylene violet (MV)] were tested on human plasma butyrylcholinesterase (BChE) and their inhibitory mechanisms were studied in detail. MV acted as a linear mixed type inhibitor of human BChE with Ki = 0.66 ± 0.06 μM and α = 13.6 ± 3.5. TBO and TH caused nonlinear inhibition of human BChE, compatible to double occupancy. Ki values estimated by nonlinear regression analysis for TBO and TH were 0.008 ± 0.003 μM and 2.1 ± 0.42 μM, respectively. The inhibitory potential of TBO was also tested on human erythrocyte AChE. TBO acted as a linear mixed type inhibitor of human AChE with Ki = 0.041 ± 0.005 μM and α = 1.6 ± 0.007. Using four site-directed BChE mutants, the role of peripheral anionic site residues of human BChE was also investigated in the binding of TBO to BChE. The peripheral anionic site mutants of BChE caused 16-69-fold increase in Ki value of TBO, compared to recombinant wild-type, suggesting that peripheral anionic site residues are involved in the binding of TBO to human BChE. In conclusion, TBO which is a potent inhibitor of human cholinesterases, may be a potential drug candidate for the treatment of Alzheimer's disease.

Keywords: Cholinesterase inhibition; Cholinesterases; Phenothiazines; Site-directed mutagenesis; Toluidine blue O.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholinesterase / metabolism*
  • Alzheimer Disease / drug therapy
  • Anions
  • Binding Sites
  • Butyrylcholinesterase / metabolism*
  • Cholinesterase Inhibitors / chemistry*
  • Dose-Response Relationship, Drug
  • Erythrocytes / enzymology
  • Humans
  • Kinetics
  • Mutagenesis, Site-Directed
  • Mutation
  • Neuroprotective Agents / chemistry
  • Phenothiazines / chemistry*
  • Protein Binding
  • Regression Analysis
  • Tolonium Chloride / chemistry*


  • Anions
  • Cholinesterase Inhibitors
  • Neuroprotective Agents
  • Phenothiazines
  • Tolonium Chloride
  • Acetylcholinesterase
  • Butyrylcholinesterase
  • thionine
  • methylene violet