Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

Abstract

Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M(-1) s(-1)), thioredoxins (k = 8.5 to 26 M(-1) s(-1)), dihydrolipoamide (k = 53 M(-1) s(-1)), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the kcat values ranging from 0.03 s(-1) to 10 s(-1) at 25 degrees C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.