title: Interactions of the antimalarial drug methylene blue with methemoglobin and heme targets in Plasmodium falciparum: a physico-biochemical study.
abstract: OBJECTIVE
  Resistance of Plasmodium falciparum to drugs has led to renewed interest of redox-active methylene blue (MB) for which no resistance has been reported so far. Moreover, MB displays unique interactions with glutathione reductase (GR). However, the mechanisms of action/interaction with potential targets of MB are yet to be elucidated. Our physico-biochemical study on MB and relevant hematin-containing targets was performed under quasi-physiological conditions.
  
  
  RESULTS
  The water deprotonation of the Fe(III)protoporphyrin dimer, the major building block of β-hematin, was studied. At pH 6, the predominant dimer possesses water coordinated to both metals. Below pH 6, spontaneous precipitation of β-hematin occurred reminiscent of hemozoin biomineralization at pH 5.0-5.5 in the food vacuole of the malarial parasite. MB also forms dimers (K(Dim)=6800 M(-1)) and firmly binds to hematin in a 2:1 hematin:MB sandwich complex (K(D)=3.16 μM). MB bioactivation catalyzed by GR induces efficient methemoglobin(Fe(III)) [metHb(Fe(III))] reduction to hemoglobin(Fe(II)). The reduction rate, mediated by leucomethylene blue (LMB), was determined (k(metHb)(red)=991 M(-1)·s(-1)) in an assay coupled to the GR/reduced form of nicotinamide adenine dinucleotide phosphate system.
  
  
  CONCLUSIONS
  Our work provides new insights into the understanding of (i) how MB interacts with hematin-containing targets, (ii) other relevant MB properties in corroboration with the distribution of the three major LMB species as a function of pH, and (iii) how this redox-active cycler induces efficient catalytic reduction of metHb(Fe(III)) to hemoglobin(Fe(II)) mediated by oxidoreductases. These physico-biochemical parameters of MB open promising perspectives for the interpretation of the pharmacology and pathophysiology of malaria and possibly new routes for antimalarial drug development.
authors.affiliation: Laboratoire de Chimie Bioorganique et Médicinale, European School of Chemistry, Polymers and Materials (ECPM), University of Strasbourg and Centre National de la Recherche Scientifique, Strasbourg, France.
authors.forename: Olga
authors.initials: O
authors.lastname: Blank
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Davioud-Charvet
authors.forename: Mourad
authors.initials: M
authors.lastname: Elhabiri
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D019281:Dimerization; D005980:Glutathione Reductase / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008706:Methemoglobin / Q000737:chemistry / Q000378:metabolism*; D008751:Methylene Blue / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D000962:Antimalarials; D006418:Heme; D009249:NADP; D008706:Methemoglobin; D005980:Glutathione Reductase; D008751:Methylene Blue