ABSTRACT
BACKGROUND: Methylene blue (MB) has been shown to be safe and effective against falciparum malaria in Africa and to have pronounced gametocytocidal properties. METHODS: Three days of treatment with artesunate (AS)-amodiaquine (AQ) combined with MB was compared with AS-AQ treatment in a randomized controlled phase IIb study; the study included 221 children aged 6-59 months with uncomplicated falciparum malaria in Burkina Faso. The primary end point was gametocyte prevalence during follow-up, as determined by microscopy and real-time quantitative nucleic acid sequence-based amplification (QT-NASBA). RESULTS: The gametocyte prevalence of Plasmodium falciparum at baseline was 3.6% (microscopy) and 97% (QT-NASBA). It was significantly lower in the AS-AQ-MB than in the AS-AQ group on day 7 of follow-up (microscopy, 1.2% vs 8.9% [P < .05]; QT-NASBA, 36.7% vs 63.3% [P < .001]). Hemoglobin values were significantly lower in the AS-AQ-MB group than in the AS-AQ group at days 2 and 7 of follow-up. Vomiting of the study medication occurred significantly more frequently in the AS-AQ-MB group. CONCLUSIONS: The combination of MB with an artemisinin-based combination therapy has been confirmed to be effective against the gametocytes of P. falciparum. MB-based combinations need to be compared with primaquine-based combinations, preferably using MB in an improved pediatric formulation. Clinical Trials Registration: NCT01407887.
Subject(s)
Amodiaquine/therapeutic use , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Malaria, Falciparum/drug therapy , Methylene Blue/therapeutic use , Amodiaquine/adverse effects , Antimalarials/adverse effects , Artemisinins/adverse effects , Artesunate , Burkina Faso , Child, Preschool , Drug Therapy, Combination/methods , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Female , Humans , Infant , Male , Methylene Blue/adverse effects , Microscopy , Plasmodium falciparum/isolation & purification , Polymerase Chain Reaction , Treatment OutcomeABSTRACT
PURPOSE: Methylene blue (MB), which was recently tested in a number of clinical malaria studies in Burkina Faso, is currently investigated for its benefit when added to artemisinin-based combination therapy. Together with a number of other antimalarials, MB is on the list of drugs which potentially induce haemolysis in patients with G6PD deficiency. Ruling out safety concerns is of major importance during drug development. METHODS: A pooled analysis was performed with patient data from four clinical studies conducted in West African children with falciparum malaria between 2003 and 2007. The primary endpoints were haemoglobin levels over time as well as haemolysis in G6PD-deficient (n = 199) and G6PD-sufficient (n = 806) children treated with MB-containing (n = 844) compared to children without MB-containing (n = 161) drug regimens. RESULTS: In the chosen model, the haemoglobin time course was significantly influenced by the G6PD genotype and the MB dose. In children with hemi- or homozygous G6PD (A-) deficiency, MB treatment with 15 mg/kg per day was associated with a significant reduction in Hb values which reached a minimum of 8.5 g/dl. Two episodes of haemolysis occurred (out of 1005 children); one in a girl heterozygous for G6PD deficiency and one in a hemizygous boy, both had received MB. CONCLUSIONS: MB treatment of malaria in Africa is associated with slightly reduced haemoglobin values in children with a full G6PD defect compared to non-G6PD deficient children. This effect appears to be of limited clinical relevance but needs to be monitored.
Subject(s)
Glucosephosphate Dehydrogenase Deficiency/blood , Hemolysis/drug effects , Malaria, Falciparum/drug therapy , Methylene Blue/adverse effects , Anemia/chemically induced , Child , Child, Preschool , Female , Hemoglobins/analysis , Humans , Infant , Male , Randomized Controlled Trials as Topic , RiskABSTRACT
Adenylate kinases (AK) play a key role in nucleotide signaling processes and energy metabolism by catalyzing the reversible conversion of ATP and AMP to 2 ADP. In the malaria parasite Plasmodium falciparum this reaction is mediated by AK1, AK2, and a GTP:AMP phosphotransferase (GAK). Here, we describe two additional adenylate kinase-like proteins: PfAKLP1, which is homologous to human AK6, and PfAKLP2. Using GFP-fusion proteins and life cell imaging, we demonstrate a cytosolic localization for PfAK1, PfAKLP1, and PfAKLP2, whereas PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane, and this localization is driven by N-myristoylation.
Subject(s)
Adenylate Kinase/metabolism , Plasmodium falciparum/enzymology , Protozoan Proteins/metabolism , Adenylate Kinase/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Cytosol/enzymology , DNA Primers/genetics , DNA, Protozoan/genetics , Humans , Intracellular Membranes/enzymology , Mitochondria/enzymology , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Vacuoles/enzymologyABSTRACT
Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage.
Subject(s)
Antimalarials/pharmacology , Glutathione Reductase/metabolism , Naphthoquinones/pharmacology , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Animals , Antimalarials/chemistry , Antimalarials/metabolism , Biocatalysis , Dose-Response Relationship, Drug , Glutathione Reductase/chemistry , Humans , Mice , Molecular Structure , Naphthoquinones/chemistry , Naphthoquinones/metabolism , Oxidation-Reduction , Parasitic Sensitivity Tests , Structure-Activity RelationshipABSTRACT
Methylene blue (MB), the first synthetic drug, has a 120-year-long history of diverse applications, both in medical treatments and as a staining reagent. In recent years there was a surge of interest in MB as an antimalarial agent and as a potential treatment of neurodegenerative disorders such as Alzheimer's disease (AD), possibly through its inhibition of the aggregation of tau protein. Here we review the history and medical applications of MB, with emphasis on recent developments.
Subject(s)
Alzheimer Disease/drug therapy , Methylene Blue/therapeutic use , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Animals , Antimalarials/chemistry , Antimalarials/therapeutic use , Humans , Methylene Blue/chemistry , tau Proteins/metabolismABSTRACT
Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPH-dependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies.
Subject(s)
Gene Silencing , Glutathione Reductase/metabolism , NADP/metabolism , Plasmodium berghei/enzymology , Plasmodium berghei/genetics , Protozoan Proteins/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Animals , Cell Proliferation , Glutathione Reductase/chemistry , Glutathione Reductase/genetics , Humans , Malaria/parasitology , Mice , Mice, Inbred C57BL , Plasmodium berghei/chemistry , Plasmodium berghei/cytology , Plasmodium berghei/growth & development , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Rats , Rats, Sprague-Dawley , Thioredoxin-Disulfide Reductase/chemistry , Thioredoxin-Disulfide Reductase/geneticsABSTRACT
OBJECTIVE: To assess the efficacy of methylene blue (MB) monotherapy in semi-immune adults with uncomplicated malaria in Burkina Faso. METHODS: In an open-label controlled phase II study with 60 semi-immune adults with uncomplicated falciparum malaria in Nouna, north-western Burkina Faso, MB monotherapy (390 mg twice daily) was given sequentially to groups of 20 adults for 7 days (MB7), 5 days (MB5) and 3 days (MB3), respectively. The primary outcome was the rate of adequate clinical and parasitological response (ACPR) on day 28 of follow-up. RESULTS: Of the study population, 27/58 (47%) and 5/51 (10%) patients still had parasites on days 2 and 3, respectively, of follow-up resulting in 9/58 (16%) early treatment failures. By day 14, no recrudescence was observed but in 4/19 (MB5) and 2/20 (MB3) individuals by day 28. The PCR-corrected rate of ACPR was 72%, 58% and 85% in groups 7, 5 and 3, respectively, by per protocol analysis. Self-limiting dysuria was the most frequent adverse event. CONCLUSIONS: MB acts slowly against the blood stages of P. falciparum. MB alone needs to be given for at least 7 days to be efficacious in the treatment of falciparum malaria but should be used in combination with a fast acting antimalarial.
Subject(s)
Antimalarials/therapeutic use , Enzyme Inhibitors/therapeutic use , Malaria, Falciparum/drug therapy , Methylene Blue/therapeutic use , Adolescent , Adult , Antimalarials/adverse effects , Burkina Faso , Drug Administration Schedule , Dysuria/etiology , Enzyme Inhibitors/adverse effects , Female , Humans , Male , Methylene Blue/adverse effects , Middle Aged , Plasmodium falciparum/isolation & purification , Polymerase Chain Reaction , Single-Blind Method , Young AdultSubject(s)
Antimalarials/therapeutic use , Artemisinins/therapeutic use , Malaria, Falciparum/drug therapy , Amodiaquine/supply & distribution , Amodiaquine/therapeutic use , Antimalarials/supply & distribution , Artemisinins/supply & distribution , Burkina Faso , Cambodia/epidemiology , Drug Combinations , Drug Resistance , Humans , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Thailand/epidemiologyABSTRACT
In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.
Subject(s)
Glutathione Reductase/deficiency , Glutathione Reductase/genetics , Malaria, Falciparum/complications , Malaria, Falciparum/enzymology , Case-Control Studies , Complement C3/metabolism , Drug Resistance/drug effects , Female , Genetic Predisposition to Disease , Glutathione/metabolism , Homozygote , Humans , Immunoglobulin G/metabolism , Inhibitory Concentration 50 , Middle Aged , Phagocytosis , Plasmodium falciparum/metabolismABSTRACT
In genome-wide screens we studied CA/C1 peptidases of malaria-causing plasmodia and their hosts (man and mouse). For Plasmodium falciparum and P. berghei, several new CA/C1 peptidase genes encoding proteases of the L- and B-family with specific promoter modules were identified. In addition, two new human CA/C1 peptidase loci and one new mouse gene locus were found; otherwise, the sets of CA/C1 peptidase genes in man and mouse seem to be complete now. In each species studied there is a multitude of CA/C1 peptidases with lysosomal localization signals and partial functional overlap according to similar but subfamily-specific structures. Individual target structures in plasmodia include residues specifically different in CA/C1 peptidase subsite 2. This is of medical interest considering CA/C1 peptidase inhibition for chemotherapy in malaria, malignancies and other diseases. Promoter structures and mRNA regulation differ widely among CA/C1 peptidase subfamilies and between mammals and plasmodia. We characterized promoter modules conserved in mouse and man for the CA/C1 peptidase families B and L (with the L-like subfamily, F-like subfamily and mouse-specific J-like subfamily). RNA motif searches revealed conserved regulatory elements such as GAIT elements; plasmodial CA/C1 peptidase mRNA elements include ARE elements and mammalian mRNAs contain 15-lox DICE elements.
Subject(s)
Computational Biology , Malaria/parasitology , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Plasmodium berghei/enzymology , Plasmodium falciparum/enzymology , Animals , Gene Expression Regulation, Enzymologic , Genetic Loci , Humans , Mice , Models, Molecular , Peptide Hydrolases/genetics , Phylogeny , Promoter Regions, Genetic/genetics , Protein Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulatory Elements, TranscriptionalABSTRACT
BACKGROUND: With the availability of new preventive and curative interventions, global malaria control has been strengthened significantly in recent years. Drugs effective in reducing malaria gametocytaemia might contribute to local elimination and possible long-term eradication. We here report on the effects of methylene blue (MB)-based malaria combination therapy on gametocytaemia during a randomised-controlled trial in Burkina Faso. METHODS: An open-label randomised controlled phase II study in 180 children aged 6-10 years with uncomplicated falciparum malaria was conducted in Nouna, north-western Burkina Faso. Children were randomised to MB-artesunate (AS), MB-amodiaquine (AQ), and AS-AQ (local standard of care). Overall follow-up was for 28 days, follow-up for gametocytaemia was for 14 days. FINDINGS: The treatment groups were similar in baseline characteristics and there was only one loss to follow-up. Compared to AS-AQ, both MB-containing regimens were associated with significantly reduced gametocyte carrier rates during follow-up days 3, 7, and 14. This effect was seen both in patients with and without P. falciparum gametocytaemia at baseline. INTERPRETATION: MB reveals pronounced gametocytocidal activity which appears to act against both existing and developing P. falciparum gametocytes. MB-based combination therapy thus has the potential to reduce transmission of P. falciparum malaria in endemic regions, which has important implications for future elimination and eradication strategies. TRIAL REGISTRATION: (ClinicalTrials.gov) NCT00354380.
Subject(s)
Antimalarials/administration & dosage , Germ Cells/drug effects , Malaria, Falciparum/drug therapy , Methylene Blue/administration & dosage , Amodiaquine/administration & dosage , Animals , Artemisinins/administration & dosage , Artesunate , Burkina Faso , Child , Drug Therapy, Combination , Humans , Methylene Blue/therapeutic use , Plasmodium falciparum/drug effects , Treatment OutcomeABSTRACT
Adenylate kinases (AK; ATP+AMP<-->2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10 U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P(1),P(5)-(diadenosine-5')-pentaphosphate (IC(50)=200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P. falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein.
Subject(s)
Acyltransferases/chemistry , Adenylate Kinase/chemistry , Isoenzymes/chemistry , Plasmodium falciparum/enzymology , Protozoan Proteins/chemistry , Acyltransferases/genetics , Acyltransferases/metabolism , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Amino Acid Sequence , Animals , Catalysis , Cloning, Molecular , Isoenzymes/genetics , Isoenzymes/metabolism , Mass Spectrometry , Molecular Sequence Data , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.
Subject(s)
Peroxidases/genetics , Plasmodium berghei/enzymology , Animals , Base Sequence , Blotting, Western , DNA Primers , Life Cycle Stages , Plasmodium berghei/growth & development , RNA, Messenger/genetics , Rats , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.
Subject(s)
Methylene Blue/pharmacokinetics , Sulfhydryl Compounds/metabolism , Trypanocidal Agents/pharmacokinetics , Trypanosoma/enzymology , Animals , Antioxidants/metabolism , Catalysis , Dihydrolipoamide Dehydrogenase/antagonists & inhibitors , Glutathione/analogs & derivatives , Glutathione/metabolism , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Spermidine/analogs & derivatives , Spermidine/metabolismABSTRACT
BACKGROUND: Besides existing artemisinin-based combination therapies, alternative safe, effective and affordable drug combinations against falciparum malaria are needed. Methylene blue (MB) was the first synthetic antimalarial drug ever used, and recent studies have been promising with regard to its revival in malaria therapy. The objective of this study was to assess the safety and efficacy of two MB-based malaria combination therapies, MB-artesunate (AS) and MB-amodiaquine (AQ), compared to the local standard of care, AS-AQ, in Burkina Faso. METHODS AND FINDINGS: Open-label randomised controlled phase II study in 180 children aged 6-10 years with uncomplicated falciparum malaria in Nouna, north-western Burkina Faso. Follow-up was for 28 days and analysis by intention-to-treat. The treatment groups were similar in baseline characteristics and there was only one loss to follow-up. No drug-related serious adverse events and no deaths occurred. MB-containing regimens were associated with mild vomiting and dysuria. No early treatment failures were observed. Parasite clearance time differed significantly among groups and was the shortest with MB-AS. By day 14, the rates of adequate clinical and parasitological response after PCR-based correction for recrudescence were 87% for MB-AS, 100% for MB-AQ (p = 0.004), and 100% for AS-AQ (p = 0.003). By day 28, the respective figure was lowest for MB-AS (62%), intermediate for the standard treatment AS-AQ (82%; p = 0.015), and highest for MB-AQ (95%; p<0.001; p = 0.03). CONCLUSIONS: MB-AQ is a promising alternative drug combination against malaria in Africa. Moreover, MB has the potential to further accelerate the rapid parasite clearance of artemisinin-based combination therapies. More than a century after the antimalarial properties of MB had been described, its role in malaria control deserves closer attention. TRIAL REGISTRATION: ClinicalTrials.gov NCT00354380.
Subject(s)
Amodiaquine/administration & dosage , Artemisinins/administration & dosage , Malaria, Falciparum/drug therapy , Methylene Blue/administration & dosage , Sesquiterpenes/administration & dosage , Artesunate , Burkina Faso , Drug Therapy, Combination , Drug-Related Side Effects and Adverse Reactions , Dysuria/chemically induced , Humans , Treatment Outcome , Vomiting/chemically inducedABSTRACT
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.
Subject(s)
Disulfides/metabolism , Methylene Blue/metabolism , Oxidoreductases/metabolism , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Protozoan Proteins/metabolism , Aerobiosis , Animals , Binding Sites , Humans , Hydrogen-Ion Concentration , Kinetics , Methylene Blue/analogs & derivatives , Methylene Blue/chemistry , Methylene Blue/pharmacology , Oxidation-Reduction , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/chemistry , Protozoan Proteins/chemistry , Substrate SpecificityABSTRACT
Hereditary glutathione reductase (GR) deficiency was found in only 2 cases when testing more than 15 000 blood samples. We have investigated the blood cells of 2 patients (1a and 1b) in a previously described family suffering from favism and cataract and of a novel patient (2) presenting with severe neonatal jaundice. Red blood cells and leukocytes of the patients in family 1 did not contain any GR activity, and the GR protein was undetectable by Western blotting. Owing to a 2246-bp deletion in the patients' DNA, translated GR is expected to lack almost the complete dimerization domain, which results in unstable and inactive enzyme. The red blood cells from patient 2 did not exhibit GR activity either, but the patient's leukocytes contained some residual activity that correlated with a weak protein expression. Patient 2 was found to be a compound heterozygote, with a premature stop codon on one allele and a substitution of glycine 330, a highly conserved residue in the superfamily of NAD(P)H-dependent disulfide reductases, into alanine on the other allele. Studies on recombinant GR G330A revealed a drastically impaired thermostability of the protein. This is the first identification of mutations in the GR gene causing clinical GR deficiency.
Subject(s)
Cataract/genetics , Favism/genetics , Genetic Diseases, Inborn/genetics , Glutathione Reductase/deficiency , Jaundice, Neonatal/genetics , Sequence Deletion , Alleles , Amino Acid Substitution , Cataract/enzymology , Child, Preschool , Codon, Nonsense/genetics , Erythrocytes/enzymology , Favism/enzymology , Female , Genetic Diseases, Inborn/enzymology , Glutathione Reductase/chemistry , Heterozygote , Humans , Infant, Newborn , Jaundice, Neonatal/enzymology , Leukocytes/enzymology , Male , Middle Aged , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
The development of safe, effective and affordable drug combinations against malaria in Africa is a public health priority. Methylene blue (MB) has a similar mode of action as chloroquine (CQ) and has moreover been shown to selectively inhibit the Plasmodium falciparum glutathione reductase. In 2004, an uncontrolled dose-finding study on the combination MB-CQ was performed in 435 young children with uncomplicated falciparum malaria in Burkina Faso (CQ monotherapy had a > 50% clinical failure rate in this area in 2003). Three serious adverse events (SAE) occurred of which one was probably attributable to the study medication. In the per protocol safety analysis, there were no dose specific effects. The overall clinical and parasitological failure rates by day 14 were 10% [95% CI (7.5%, 14.0%)] and 24% [95% CI (19.4%, 28.3%)], respectively. MB appears to have efficacy against malaria, but the combination of CQ-MB is clearly not effective in the treatment of malaria in Africa.
Subject(s)
Chloroquine/therapeutic use , Malaria, Falciparum/drug therapy , Methylene Blue/administration & dosage , Methylene Blue/therapeutic use , Burkina Faso/epidemiology , Child, Preschool , Chloroquine/administration & dosage , Chloroquine/adverse effects , Dose-Response Relationship, Drug , Drug Therapy, Combination , Female , Humans , Infant , Malaria, Falciparum/epidemiology , Male , Methylene Blue/adverse effects , Random Allocation , Treatment FailureABSTRACT
4',5'-Dibromo-2',7'-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC(50)) of 180 microM. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC(50) of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.
Subject(s)
Antimalarials/pharmacology , Fluoresceins/pharmacology , Plasmodium falciparum/drug effects , Animals , Antimalarials/pharmacokinetics , Cell Line , Drug Resistance , Eosine I Bluish , Fibroblasts/parasitology , Fluoresceins/pharmacokinetics , Glutathione Reductase/antagonists & inhibitors , Glutathione Reductase/metabolism , Humans , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Plasmodium falciparum/enzymology , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Tetrahydrofolate Dehydrogenase/metabolism , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Thioredoxin-Disulfide Reductase/metabolism , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/metabolismABSTRACT
Glutathione reductase is an important housekeeping enzyme for redox homeostasis both in human cells and in the causative agent of tropical malaria, Plasmodium falciparum. Glutathione reductase inhibitors were shown to have anticancer and antimalarial activity per se and to contribute to the reversal of drug resistance. The development of menadione chemistry has led to the selection of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called M(5), as a potent reversible and uncompetitive inhibitor of both human and P. falciparum glutathione reductases. Here we describe the synthesis and kinetic characterization of a fluoromethyl-M(5) analogue that acts as a mechanism-based inhibitor of both enzymes. In the course of enzymatic catalysis, the suicide substrate is activated by one- or two-electron reduction, and then a highly reactive quinone methide is generated upon elimination of the fluorine. Accordingly the human enzyme was found to be irreversibly inactivated with a k(inact) value of 0.4 +/- 0.2 min(-1). The crystal structure of the alkylated enzyme was solved at 1.7 A resolution. It showed the inhibitor to bind covalently to the active site Cys58 and to interact noncovalently with His467', Arg347, Arg37, and Tyr114. On the basis of the crystal structure of the inactivated human enzyme and stopped-flow kinetic studies with two- and four-electron-reduced forms of the unreacted P. falciparum enzyme, a mechanism is proposed which explains naphthoquinone reduction at the flavin of glutathione reductase.