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 k cat values ranging from 0.03 s ؊1 to 10 s ؊1 at 25°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 Cys 58 /Cys 63 . The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H 2 O 2 at the expense of O 2 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 cellpathological and clinical contexts.
Methylene blue (MB or MBϩ ) [also known as methylthionine hydrochloride or 3,7-bis(dimethylamino)phenothiazin-5-ium chloride] was the very first synthetic compound to be used as a drug. Paul Ehrlich, who introduced the concept of modern target-based chemotherapy using MB as an example, and Paul Guttmann described the compound as being an effective antimalarial agent (28). Despite its beneficial antimalarial activity, the drug disappeared from the scene because up-and-coming compounds such as chloroquine were more effective; in addition, soldiers resented taking MB because of inevitable but harmless side effects: green or blue urine and bluish sclerae (47, 55).After MB was revisited as an antimalarial agent (6, 53) and found to be an inhibitor of Plasmodium falciparum glutathione (GSH) reductase (GR) (23), it was studied as a partner drug in antimalarial drug combinations (2,39,48,51).Compared with other antiparasitic agents, MB is affordable and registered in most countries (as the treatment of choice for acute and chronic methemoglobinemia [16,17,36]), and it can be made internationally available in sufficient dosages (51). The price for treating a malaria episode in a child with an MB-containing drug combination would be less than €0.50 (40). Drug resistance has not been reported for MB and could not be provoked in rodent malaria models (52, 53).Because of its favorable properties, including the differential staining of cell biological structures and protein cr...
