Interactions of the antimalarial drug methylene blue with methemoglobin and heme targets in Plasmodium falciparum: A physico-biochemical study

摘要

Aims: 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. Innovation and Conclusion: 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.