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 (KDim=6800 M−1) and firmly binds to hematin in a 2:1 hematin:MB sandwich complex (KD=3.16 μM). MB bioactivation catalyzed by GR induces efficient methemoglobin(FeIII) [metHb(FeIII)] reduction to hemoglobin(FeII). The reduction rate, mediated by leucomethylene blue (LMB), was determined (kmetHbred=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(FeIII) to hemoglobin(FeII) 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. Antioxid. Redox Signal. 17, 544–554.
Get full access to this article
View all access options for this article.
References
1.
AidooM, TerlouwDJ, KolczakMS, McElroyPD, ter KuileFO, KariukiS, NahlenBL, LalAA, UdhayakumarV. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet, 359:1311–1312. 2002.
2.
AkoachereM, BuchholzK, FischerE, BurhenneJ, HaefeliWE, SchirmerRH, BeckerK. In vitro assessment of methylene blue on chloroquine-sensitive and -resistant Plasmodium falciparum strains reveals synergistic action with artemisinins. Antimicrob Agents Chemother, 49:4592–4597. 2005.
3.
AnsteyNM, HassanaliMY, MlalasiJ, ManyengaD, MwaikamboED. Elevated levels of methaemoglobin in Tanzanian children with severe and uncomplicated malaria. Trans R Soc Trop Med Hyg, 90:147–151. 1996.
4.
AsherC, de VilliersKA, EganTJ. Speciation of ferriprotoporphyrin IX in aqueous and mixed aqueous solution is controlled by solvent identity, pH, and salt concentration. Inorg Chem, 48:7994–8003. 2009.
5.
AyiK, TurriniF, PigaA, AreseP. Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait. Blood, 104:3364–3371. 2004.
6.
BauerH, Fritz-WolfK, WinzerA, KühnerS, LittleS, YardleyV, VezinH, PalfeyB, SchirmerRH, Davioud-CharvetE. A fluoro analogue of the menadione derivative 6-[2′-(3′-methyl)-1′,4′-naphthoquinolyl]hexanoic acid is a suicide substrate of glutathione reductase. Crystal structure of the alkylated human enzyme. J Am Chem Soc, 128:10784–10794. 2006.
7.
BeutlerE, BaludaMC. Methemoglobin reduction studies of the interaction between cell populations and of the role of methylene blue. Blood, 22:323–333. 1963.
8.
BraswellE. Evidence for trimerization in aqueous solutions of methylene blue. J Phys Chem, 72:2477–2483. 1968.
9.
BuchholzK, CominiMA, WissenbachD, SchirmerRH, Krauth-SiegelRL, GromerS. Cytotoxic interactions of methylene blue with trypanosomatid-specific disulfide reductases and their dithiol products. Mol Biochem Parasitol, 160:65–69. 2008.
10.
BuchholzK, SchirmerRH, EubelJK, AkoachereMB, DandekarT, BeckerK, GromerS. Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum. Antimicrob Agents Chemother, 52:183–191. 2008.
11.
CappadoroM, GiribaldiG, O'BrienE, TurriniF, MannuF, UlliersD, SimulaG, LuzzattoL, AreseP. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency. Blood, 92:2527–2534. 1998.
CoulibalyB, ZoungranaA, MockenhauptFP, SchirmerRH, KloseC, MansmannU, MeissnerPE, MüllerO. Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomised controlled trial. PLoS One, 4:e5318. 2009.
14.
CrespoMP, TilleyL, KlonisN. Solution behavior of hematin under acidic conditions and implications for its interactions with chloroquine. J Biol Inorg Chem, 15:1009–1022. 2010.
Davioud-CharvetE, LanfranchiDA. Subversive substrates of glutathione reductases from P. falciparum-infected red blood cells as antimalarial agents. Apicomplexan Parasites—Molecular Approaches Toward Targeted Drug Development, 2BeckerKfrom the series Drug Discovery in Infectious DiseasesSelzerPM. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2011ISBN 978-3-527-32731-7.
17.
De VilliersKA, KaschulaCH, EganTJ, MarquesHM. Speciation and structure of ferriprotoporphyrin IX in aqueous solution: spectroscopic and diffusion measurements demonstrate dimerization, but not μ-oxo dimer formation. J Biol Inorg Chem, 12:101–117. 2007.
18.
DietrichLE, Price-WhelanA, PetersenA, WhiteleyM, NewmanDK. The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol, 61:1308–1321. 2006.
19.
EatonWA, HansonLK, StephensPJ, SutherlandJC, DunnJBR. Optical spectra of oxy- and deoxyhemoglobin. J Am Chem Soc, 100:4991–5003. 1978.
20.
EganTJ. Haemozoin (malaria pigment): a unique crystalline drug target. Targets, 2:115–124. 2003.
21.
EganTJ, NcokasiKK. Quinoline antimalarials decrease the rate of β-hematin formation. J Inorg Biochem, 99:1532–1539. 2005.
22.
FärberPM, ArscottLD, WilliamsCHJr., BeckerK, SchirmerRH. Recombinant Plasmodium falciparum glutathione reductase is inhibited by the antimalarial dye methylene blue. FEBS Lett, 422:311–314. 1998.
23.
GalloV, SchwarzerE, RahlfsS, SchirmerRH, van ZwietenR, RoosD, AreseP, BeckerK. Inherited glutathione reductase deficiency and Plasmodium falciparum malaria—a case study. PLoS One, 4:e7303. 2009.
24.
GeorgesJ. Deviations from Beer's law due to dimerization equilibria: theoretical comparison of absorbance, fluorescence and thermal lens measurements. Spectrochim Acta A, 51:985–994. 1995.
25.
GreenMD, XiaoL, LalAA. Formation of hydroxyeicosatetraenoic acids from hemozoin-catalyzed oxidation of arachidonic acid. Mol Biochem Parasitol, 83:183–188. 1996.
26.
GuttmanP, EhrlichP. Über die wirkung des Methylenblau bei Malaria. Berl Klin Wochenschr, 39:953–956. 1891.
27.
HaynesRK, CheuKW, TangMM, ChenMJ, GuoZF, GuoZH, CoghiP, MontiD. Reactions of antimalarial peroxides with each of leucomethylene blue and dihydroflavins: flavin reductase and the cofactor model exemplified. ChemMedChem, 6:279–291. 2011.
28.
HoggT, NagarajanK, HerzbergS, ChenL, ShenX, JiangH, WeckeM, BlohmkeC, HilgenfeldR, SchmidtCL. Structural and functional characterization of Falcipain-2, a hemoglobinase from the malarial parasite Plasmodium falciparum. J Biol Chem, 281:25425–25437. 2006.
29.
ImpertO, KatafiasA, KitaP, MillsA, Pietkiewicz-GraczykA, WrzeszczG. Kinetics and mechanism of a fast leuco-methylene blue oxidation by copper(II)—halide species in acidic aqueous media. Dalton Trans, 3:348–353. 2003.
30.
KasoziD, GromerS, AdlerH, ZocherK, RahlfsS, WittlinS, Fritz-WolfK, SchirmerRH, BeckerK. The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparison with its thioanalog methylene blue. Redox Rep, 16:154–165. 2011.
31.
KlonisN, DilanianR, HanssenE, DarmaninC, StreltsovV, DeedS, QuineyH, TilleyL. Hematin-hematin self-association states involved in the formation and reactivity of the malaria parasite pigment, hemozoin. Biochemistry, 49:6804–6811. 2010.
32.
Krauth-SiegelRL, BauerH, SchirmerRH. Dithiol proteins as guardians of the intracellular redox milieu in parasites: old and new drug targets in trypanosomes and malaria-causing plasmodia. Angew Chem Int Ed, 44:690–715. 2005.
33.
LehaneAM, HaywardR, SalibaKJ, KirkK. A verapamil-sensitive chloroquine-associated H+ leak from the digestive vacuole in chloroquine-resistant malaria parasites. J Cell Sci, 121:1624–1632. 2008.
34.
LuzzattoL. About hemoglobins, G6PD and parasites in red cells. Experientia, 51:206–208. 1995.
35.
McMillanPJ, StimmlerLM, FothBJ, McFaddenGI, MüllerS. The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases. Mol Microbiol, 55:27–38. 2005.
36.
MayJM, QuZ-C, CobbCE. Reduction and uptake of methylene blue by human erythrocytes. Am J Physiol Cell Physiol, 286:C1390–C1398. 2004.
37.
MichaelisL, GranickS. Metachromasy of basic dyestuffs. J Am Chem Soc, 67:1212–1219. 1945.
38.
MonkPMS, HodgkinsonNM, RamzanSA. Spin pairing (‘dimerisation’) of the viologen radical cation: kinetics and equilibria. Dyes Pigments, 43:207–217. 1999.
39.
MontiD, VodopivecB, BasilicoN, OlliaroP, TaramelliD. A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites. Biochemistry, 38:8858–8863. 1999.
40.
MukerjeeP, GhoshAK. Thermodynamic aspects of the self-association and hydrophobic bonding of methylene blue. A model system for stacking interactions. J Am Chem Soc, 92:6419–6424. 1970.
41.
MüllerT, JohannL, JannackB, BrücknerM, LanfranchiDA, BauerH, SanchezC, YardleyV, DeregnaucourtC, SchrévelJ, LanzerM, SchirmerRH, Davioud-CharvetE. Glutathione reductase-catalyzed cascade of redox reactions to bioactivate potent antimalarial 1,4-naphthoquinones—a new strategy to combat malarial parasites. J Am Chem Soc, 133:11557–11571. 2011.
42.
OliveiraMF, TimmBL, MachadoEA, MirandaK, AttiasM, SilvaJR, Dansa-PetretskiM, de OliveiraMA, de SouzaW, PinhalNM, SousaJJ, VugmanNV, OliveiraPL. On the pro-oxidant effects of haemozoin. FEBS Lett, 512:139–144. 2002.
43.
OzM, LorkeDE, HasanM, PetroianuGA. Cellular and molecular actions of methylene blue in the nervous system. Med Res Rev, 31:93–117. 2011.
44.
PagolaS, StephensPW, BohleDS, KosarAD, MadsenSK. The structure of malaria pigment β-haematin. Nature, 404:307–310. 2000.
45.
PatilK, PawarR, TalapP. Self-aggregation of methylene blue in aqueous medium and aqueous solutions of Bu4NBr and urea. Phys Chem Chem Phys, 2:4313–4317. 2000.
Price-WhelanA, DietrichLE, NewmanDK. Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14. J Bacteriol, 189:6372–6381. 2007.
48.
RomeroD, TraxlerMF, LópezD, KolterR. Antibiotics as signal molecules. Chem Rev, 111:5492–5505. 2011.
49.
RoweA, ChuhKN, LubinAA, MillerEA, CookBM, HollisDN, PlaxcoKW. Electrochemical biosensors employing an internal electrode attachment site achieve reversible, high gain detection of specific nucleic acid sequences. Anal Chem[Epub ahead of print]DOI:10.1021/ac202171×2011.
SchirmerRH, MüllerJG, Krauth-SiegelRL. Disulfide reductase inhibitors as chemotherapeutic agents: the design of drugs for trypanosomiasis and malaria. Angew Chem Int Ed, 34:141–154. 1995.
53.
SchwarzerE, TurriniF, UlliersD, GiribaldiG, GinsburgH, AreseP. Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment. J Exp Med, 176:1033–1041. 1992.
54.
SchwarzerE, BellomoG, GiribaldiG, UlliersD, AreseP. Phagocytosis of malarial pigment haemozoin by human monocytes: a confocal microscopy study. Parasitology, 123:125–131. 2001.
55.
SinghalGS, RabinowitchE. Changes in the absoption spectrum of methylene blue with pH. J Phys Chem, 71:3347–3349. 1967.
56.
SlaterAF, SwiggardWJ, OrtonBR, FlitterWD, GoldbergDE, CeramiA, HendersonGB. An iron-carboxylate bond links the heme units of malaria pigment. Proc Natl Acad Sci U S A, 88:325–329. 1991.
57.
SteeleCW, SpinkWW. Methylene blue in the treatment of poisonings associated with methemoglobinemia. New England J Med, 208:1152–1153. 1933.
58.
ThurstonJP. The chemotherapy of Plasmodium berghei. I. Resistance to drugs. Parasitology, 43:246–252. 1953.
59.
XiaoY, LubinAA, BakerBR, PlaxcoKW, HeegerAJ. Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex. Proc Natl Acad Sci U S A, 103:16677–16680. 2006.
60.
YimG, WangHH, DaviesJ. Antibiotics as signalling molecules. Philos Trans R Soc Lond B Biol Sci, 362:1195–1200. 2007.
61.
YoshidaS, IizukaT, NozawaT, HatanoM. Studies on the charge transfer band in high spin state of ferric myoglobin and hemoglobin by low temperature optical and magnetic circular dichroism spectroscopy. Biochim Biophys Acta Protein Structure, 405:122–135. 1975.
62.
ZoungranaA, CoulibalyB, SiéA, Walter-SackI, MockenhauptFP, KouyatéB, SchirmerRH, KloseC, MansmannU, MeissnerP, MüllerO. Safety and efficacy of methylene blue combined with artesunate or amodiaquine for uncomplicated falciparum malaria: a randomized controlled trial from Burkina Faso. PLoS One, 3:e1630. 2008.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
