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The Glyoxalase System, Inhibition of Thioredoxin Reductase and Use of Methylene Blue as Drug Development Strategies against the Malarial Parasite Plasmodium falciparum A thesis submitted in fulfilment of the German degree doctor rerum naturalium (Dr. rer. nat.) Faculty of Biology and Chemistry (FB 08) Justus-Liebig-University, Giessen, Germany Monique B. Akoachere The work reported in this thesis was carried out during the period of April 2002 to September 2005 at the Institute of Nutritional Biochemistry, Interdisciplinary Research Centre, Justus-Liebig-University, Giessen, Germany. The work was supported by the German Academic Exchange Service (DAAD) and supervised by Prof. Dr. med. Katja Becker-Brandenburg and Prof. Dr. Albrecht Bindereif. Prof. Dr. Juergen Mayer Faculty of Biology and Chemistry Justus-Liebig-University Giessen Karl-Glöckner-Strasse 21, 35394 Giessen Prof. Dr. Albrecht Bindereif Faculty of Biology and Chemistry Justus-Liebig-University Giessen Heinrich-Buff-Ring 58, 35392 Giessen Prof. Dr. med. Katja Becker-Brandenburg Faculty of Agricultural and Nutritional Sciences, Home Economics and Enviromental Management Justus-Liebig-University Giessen Heinrich-Buff-Ring 26-32, 35392 Giessen Additional Jury Member: Prof. Dr. Rudolf Geyer Justus-Liebig-University Giessen Friedrichstrasse 24, 35392 Giessen Berichte aus der Biochemie Monique B. Akoachere
The Glyoxalase System, Inhibition of Thioredoxin
Reductase and Use of Methylene Blue as Drug
Development Strategies against the Malarial
Parasite Plasmodium falciparum
D 26 (Diss. Universität Giessen) Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes Bibliographic information published by Die Deutsche Bibliothek
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Zugl.: Giessen, Univ., Diss., 2005 Copyright Shaker Verlag 2005All rights reserved. No part of this publication may be reproduced, stored in aretrieval system, or transmitted, in any form or by any means, electronic,mechanical, photocopying, recording or otherwise, without the prior permissionof the publishers.
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ISBN 3-8322-4687-8ISSN 1434-5536 Shaker Verlag GmbH • P.O. BOX 101818 • D-52018 AachenPhone: 0049/2407/9596-0 • Telefax: 0049/2407/9596-9Internet: • eMail: [email protected] This thesis is the original work of Akoachere Monique Bate. Other sources of information have been properly quoted. The work has not been used to obtain any other university Acknowledgements To begin, I would like to say that I am extremely thankful to Professors Albrecht Bindereif and Katja Becker-Brandenburg for not only giving me the opportunity to carry out my PhD project at the University of Giessen, Germany, but also for supervising and standing by the project during the entire course. To Katja, I wish to say thank you for your continuous scientific and personal encouragement especially in difficult times. Special thanks go to Prof. Heiner Schirmer for his support and collaboration throughout the PhD project. I would also like to thank the "Deutscher Akademischer Austauschdienst" (DAAD) for the unrelentless financial support throughout the PhD project and during the German language course at the Goethe Institute of Göttingen. I would also like to thank the entire working group of Professor Becker-Brandenburg for the friendly working environment. To Stefan Rahlfs and Rimma Iozef, I will say thank you for your support and collaboration on the glyoxalase system. Elisabeth, I do appreciate the training I got from you in the cell culture laboratory. To Xu Ying and Taiwo Ojurongbe, I say thank you for helping to carry out statistical analysis of the results obtained from drug combination assays. To the other PhD colleagues in the lab, Julia Bolt-Ulschmidt, Christine Nickel, Sabine Urig, Marcel Deponte, Sasa Koncarevic, Kathrin Buchholz and Boniface Mailu, I say thanks for all the ideas I have gained from you during our numerous discussions. To the other co-workers of the group, Tammy, Ulli, Beate, Marina, Marita, Johanna, Simone, Nicole, Annette and Doris, I really do appreciate whatever help – in one way or the other – I received from you. I would like to thank the entire Akoachere family for their encouragement with respect to the PhD project. Special thanks go to my brother Ashu Akoachere for helping me find my supervisor Katja and also for his struggle concerning the DAAD scholarship. To my Dad, E. B. Akoachere, whose dream had always been that I become a "doctor" right from the time I was still a kid, I am happy that I have been able to fulfil this dream of yours while you are still alive. To my Mum, Felicia Akoachere, I wish to thank you for your steadfast prayers and kindly support for all your kids. To my other brethren, Alfred, George, Johnson, Barbara, Oben, Nkongho, Arrey, Ayuk, Paula, Julliet and Kate, I say thanks for being there for me. To friends (Sylvia, Adrienne), I say thanks for the encouragement you gave me in all aspects of life. To wellwishers most of whom I got to know during my stay in Giessen, thank you all for the good times we shared. Last and most important, my sincere appreciation goes to God Almighty for His continuous love, protection and guidance over me. Original Publications 1. Andricopulo, A. D., Nickel, C., Krogh, R., Akoachere, M., McLeish, M. J., Davioud- Charvet, E., Kenyon, G. L., Arscott, D. L., Williams, C. H. Jr. and Becker, K. (2005). Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents. Submitted to Bioorganic and Medicinal Chemistry Letters. 2. Andricopulo, A. D., Akoachere, M., Krogh, R., Nickel, C., McLeish, M. J., Davioud- Charvet, E., Kenyon, G. L., Arscott, D. L., Williams, C. H. Jr. and Becker, K. (2005). Thioredoxin reductase of the malarial parasite Plasmodium falciparum – Inhibitor development as a basis for novel chemotherapeutic strategies. Flavins and Flavoproteins in press. 3. Akoachere, M., Buchholz, K., Fischer, E., Burhenne, J., Haefeli, W., Schirmer, H. and Becker, K. (2005). In vitro assessment of methylene blue on chloroquine sensitive and resistant Plasmodium falciparum strains reveals synergistic action with artemisinins. Antimicrobial Agents and Chemotherapy 49: 4592-4597. 4. Akoachere, M., Iozef, R., Rahlfs, S., Deponte, M., Mannervik, B., Creighton, D. J., Schirmer, H., Becker, K. (2005) Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts. Biological Chemistry, 386: 41-52. 5. Cho-Ngwa, F., Akoachere, M. and Titanji, V. P. (2003). Sensitive and specific serodiagnosis of riverblindness using Onchocerca ochengi antigens. Acta Tropica 89: 25-32. Abstracts in Meetings • Akoachere, M., Buchholz, K., Fischer, E., Burhenne, J., Haefeli, W., Schirmer, H. and Becker, K. (2005). Methylene blue in antimalarial drug combinations. In vitro effects on Plasmodium falciparum strains. 54th Annual Meeting of the American Society of Tropical Medicine and Hygiene (ASTMH), Washington D. C., USA. • Akoachere, M., Iozef, R., Rahlfs, S., Deponte, M., Schirmer, R. H. and Becker, K. (2005). Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts. Drug development seminar, Bernhardt Nocht Institute, Hamburg. • Andricopulo, A. D., Nickel, C., Krogh, R., Akoachere, M., McLeish, M. J., Davioud-Charvet, E., Kenyon, G. L., Arscott, D. L., Williams, C. H. Jr. and Becker, K. (2005). Novel inhibitors of the thioredoxin reductase from Plasmodium falciparum as potential antimalarial agents. 15th International Symposium on Flavins and Flavoproteins, Shonan Village, Japan. • Akoachere, M. B., Iozef, R., Rahlfs, S., Deponte, M., Schirmer, R. H. and Becker, K. (2005). Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparism with their human counterparts. First Annual BiolMalPar Conference Meeting, EMBL, Heidelberg. • Rahlfs, S., Iozef, R., Akoachere, M., Schirmer, R. H. and Becker, K. (2004). Glyoxalase I of the malarial parasite Plasmodium falciparum: Evidence for subunit fusion. Jahrestagung der Deutschen Gesellschaft für Parasitologie, Würzburg. • Akoachere, M., Iozef, R., Rahlfs, S., and Becker, K. (2003). The glyoxalase system of the malarial parasite Plasmodium falciparum. MBML (Molecular Biology and Medicine of the Lung) Retreat Program, Marburg. Table of Contents Table of Contents 1 Introduction and Rationale . 1 1.1.1 The Anopheles vector . 2 1.1.2 Life cycle of the parasite . 3 1.1.3 Pathophysiology of the disease . 4 1.1.4 Diagnosis of malaria. 4 1.1.5 Control . 6 Control through prevention of transmission. 6 Control through therapy . 7 Control through vaccines. 11 1.2 Rationale of the study. 13 1.2.1 The glyoxalase system. 13 Non-glyoxalase metabolism of methylglyoxal. 17 1.2.2 The thioredoxin reductase system . 19 1.2.3 Methylene blue in antimalarial therapy . 20 1.2.4 Other aspects of the PhD project . 23 Glutathionylation of thioredoxin of P. falciparum. 23 Lipoamide dehydrogenases of P. falciparum . 24 1.3 Objectives of the study . 25 1.3.1 The glyoxalase system. 25 1.3.2 The thioredoxin reductase of P. falciparum . 25 Table of Contents 1.3.3 Methylene blue in antimalarial chemotherapy . 25 2.1 Materials. 26 2.1.1 Chemicals . 26 2.1.2 Malaria drugs . 26 2.1.3 Enzymes . 26 2.1.4 Antibodies. 27 2.1.6 Instruments . 27 2.1.7 Biological materials . 28 cDNA libraries and erythrocytes . 28 Plasmids. 28 Escherichia coli cells. 28 Strains of P. falciparum. 29 2.1.8 Buffers and solutions . 29 Solutions for DNA electrophoresis. 29 Solutions for protein electrophoresis. 29 Solutions for western blotting. 30 Extraction of parasites from infected red blood cells . 31 2.1.9 Growth medium. 31 identification. 32 3.3 Cloning and overexpression of genes. 33 3.4 Site-directed mutagenesis . 34 3.5 Purification of glyoxalase enzymes . 34 3.6 SDS-PAGE . 37 3.7 Western blotting . 37 3.8 Concentration of proteins . 38 3.9 Determination of protein concentrations . 38 3.9.1 A280 method . 38 3.9.2 Bradford method. 39 3.10 Enzymatic assays . 39 3.11 Inhibition studies . 41 Table of Contents 3.12 Glutathionylation assays . 42 3.13 Metal ion analysis . 42 3.14 Structure prediction of glyoxalase enzymes . 42 3.15 Protein crystallization . 43 3.15.1 Crystallization screening . 43 3.15.2 Crystallization optimization . 44 3.16 Cell culture experiments . 45 3.16.1 Synchronization . 45 3.16.2 Studies on MB uptake . 46 3.16.3 Determination of enzymatic activity from parasitic extracts. 46 3.16.4 Drug susceptibility tests . 47 WHO microtest. 47 Semi-automated microdilution method . 49 Drug combination assays. 51 3.16.5 Statistics. 52 4.1 The glyoxalase system.53 4.1.1 Recombinant production of P. falciparum glyoxalases . 53 4.1.2 Kinetic characterization. 56 4.1.3 Inhibition studies . 58 4.1.4 Metal ion analysis. 60 4.1.5 Cell culture experiments. 60 4.1.6 Crystallization experiments . 61 4.1.7 Plasmodium falciparum glyoxalase structure predictions. 62 4.2 The inhibition of PfTrxR . 67 4.2.1 Kinetic analyses on isolated enzymes . 67 4.2.2 Effects of the inhibitors on P. falciparum in culture . 68 in vitro antimalarial effects of MB. 69 4.3.1 Stage-specificity of MB action . 69 4.3.2 Studies on MB uptake . 71 4.3.3 Effects of MB upon combination with other drugs . 71 Combination of MB with clinically-used antimalarials . 71 Combination of MB with artemisinins . 72 characterization of PfLipDHs. 77 Table of Contents 4.5 Glutathionylation of PfTrx . 77 5 Discussion . 80 5.1 The glyoxalase system . 81 5.2 The thioredoxin system . 84 5.3 Methylene blue in antimalarial chemotherapy . 86 5.4 Glutathionylation of PfTrx . 90 6 References. 91 6.2 Websites. 102 Table of Contents Figure 1: Geographic distribution of malaria around the world. 1 Figure 2: Life cycle of the parasite Plasmodium falciparum. 4 Figure 3: Blood stages of P. falciparum (Malaria Manual, 2003). 5 Figure 4: Diagram of P. falciparum trophozoite residing in an erythrocyte. 7 Figure 5: Structures of selected antimalarial drugs. 9 Figure 6: Malaria life cycle and vaccine targets. 12 Figure 7: Reactions of the glyoxalase system. 14 Figure 8: Physiological formation of 2-oxoaldehydes. 16 Figure 9: Chemical development of antimalarial drugs from methylene blue. 21 Figure 10: Structure of the S-(N-hydroxy-N-arylcarbamoyl)glutathiones. 42 Figure 11: Diagram of hanging drop method. 44 Figure 12: Semi-automated microdilution method: Arrangement of drugs on a 96 well Figure 13: Alignment of N- and C-terminal halves of P. falciparum glyoxalase I with human glyoxalase I. 54 Figure 14: Alignment of glyoxalases II. 55 Figure 15: SDS-PAGE of active recombinantly produced P. falciparum glyoxalases. 56 Figure 16: Dixon plots comparing competitive inhibition of (A) HBPC-GSH and (B) S- p-bromobenzylglutathione on P. falciparum glyoxalase I using the methylglyoxal-GSH adduct as substrate. 59 Figure 17: Crystals of cGloI. 62 Figure 18: Model of the active sites and hydrophobic binding pocket of GloI based on the crystal structure of human GloI (Cameron et al., 1999a). 64 Figure 19: Model of tGloII based on the crystal structure of human GloII.….65 Figure 20: Model of the metal binding site of tGloII and cGloII…………………….…66 Figure 21: Model of the glutathione-binding site of tGloII and cGloII……………….66 Figure 22: Cornish-Bowden plot showing that compound 3 inhibits PfTrxR uncompetitively with respect to PfTrx. 68 Figure 23: Convex isobologram indicating the antagonistic effects of compound 1 upon combination with chloroquine, methylene blue and artemisinin. 69 Table of Contents Figure 24: Stage specificity of methylene blue action on the CQ resistant P. falciparum Figure 25: FIC50 (left) and FIC90-values (right) of MB and CQ determined at various dosage ratios (1:1, 1:3, and 3:1) and in independent experiments (indicated by the lines) on CQ-resistant (K1) and CQ-sensitive (3D7) strains of P. Figure 26: FIC50 (left) and FIC90 values (right) of MB and artemisinin at various fixed dosage ratios on different P. falciparum strains. 75 Figure 27: Amino acid sequence of recombinant hexa-histidyl-tagged PfTrx indicating the two peptide fragments spanning the glutathionylated Cys54 (red) of Figure 28: MALDI-TOF mass spectra of PfTrx peptides. 79 Figure 29: Factors affecting parasite resistance. 81 Table of Contents Table 1: Factors contributing to development and spread of drug resistance . 11 Table 2: Cloning and overexpression of human and P. falciparum glyoxalase genes.35 Table 3: Purification of P. falciparum and human glyoxalases. 36 Table 4: Optimization of the expression conditions for PfLipDH1 and PfLipDH2. 36 Table 5: Characteristics of Plasmodium falciparum strains employed . 45 Table 6: Kinetic properties of P. falciparum and human glyoxalases I . 57 Table 7: Kinetic properties of P. falciparum and human glyoxalases II . 57 Table 8: IC50 and Ki values of S-(N-aryl-N-hydroxycarbamoyl)glutathiones on human and P. falciparum glyoxalases. 58 Table 9: Structures of the most potent PfTrxR inhibitors; compounds 1-3 and corresponding IC50 values on P. falciparum TrxR and human TrxR. 67 Table 10: Clearance of the medium from MB by P. falciparum parasitized erythrocytes Table 11: Effects of combination of MB with clinically-used antimalarials. 73 Table 12 : In vitro drug combination assays of the artemisinins, piperaquine and chloroquine with MB on P. falciparum. 76 Table of Contents Scheme 1: Structure of methylene blue. 20 Scheme 2: The lipoamide dehydrogenase reaction . 24 Absorption at . nm Artemisinin-based combination therapies To give a concentration of; to give a volume of Advanced glycation endproducts Ammonium persulphate Methylene blue + chloroquine drug combination Bovine serum albumin Counts per minute  Da Dalton  DDT Dimethylsulfoxide Deoxyribonucleic acid  dNTP Deoxyribonucleotide triphosphate  DTE Dithioerythritol  DTNB Dithionitrobenzene Ethylenediaminetetraacetic acid Flavin adenine dinucleotide Fractional inhibitory concentration Glucose-6-phosphate dehydrogenase Glyoxalase I-like protein Glutathione reductase  GSH/GSSG Glutathione (reduced /oxidized)  GST  HCPC-GSH S-(N-hydroxy-N-chlorophenylcarbamoyl)glutathione  HIV/AIDS Human immunodeficiency virus / Acquired immune deficiency Histidine rich protein  IC Inhibitory concentration  IPTG Insecticide treated nets  LC/MS/MS Liquid chromatography / Mass spectrometry / Mass spectrometry  LDH Lactate dehydrogenase  MALDI-TOF Matrix-assisted laser desorption ionization – Time of flight  MB 4-Morpholinopropane sulfonic buffer Merozoite surface protein  NADH/NAD+ Reduced /oxidized nicotinamide adenine dinucleotide  NADPH/NADP+ Reduced /oxidized nicotinamide adenine dinucleotide phosphate  NPRBC Non-parasitized red blood cells Nucleotide triphosphates Polymerase chain reaction Polyethylene glycol Plasmodium falciparum Pentose phosphate pathway Parasitized red blood cells Ring-infected erythrocyte surface antigen Ribonucleic acid Rounds per minute S-D-lactoylglutathione Sodium dodecyl sulphate  SDS-PAGE Sodium dodecyl sulphate – polyacrylamide gel electrophoresis  TEMED Thioredoxin reductase Unit of enzyme activity (µmol/min) World Health Organisation Malaria is a disease caused by protozoan parasites of the genus Plasmodium and is responsible for about half a billion diseases cases and 2-3 million deaths each year. Much of the parasite's success to establish persistent infections is attributed to evasion of the human immune defense system through antigenic variation and increasing development of resistance to all currently available antimalarial drugs except the artemisinins. The difference in structure and mode of action of the artemisinins underlines the fact that new antimalarial drugs – with differential modes of action – are an urgent priority in order to circumvent plasmodial resistance mechanisms in the absence of effective vaccines or vector control measures. By means of rational drug design and re-evaluation of an ancient antimalarial drug, three new drug development strategies against the deadliest malarial parasite, Plasmodium falciparum, were developed within the frame of this thesis in order to design possible new mechanism drugs and prevent resistance development to artemisinin. First, a complete functional glutathione-dependent glyoxalase (Glo) detoxification system – comprising a cytosolic GloI (cGloI), a GloI-like protein (GILP) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) – was characterized in direct comparison with the isofunctional human host enzymes. Kinetic and structural similarities of enzymes of both systems were described; however, striking differences – especially for the GloIs – were also detected which could be exploited for drug development. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were found to be active in the lower nanomolar range and could be used as lead structures in the development of more selective inhibitors of the P. falciparum glyoxalase system (Akoachere et al., 2005). Secondly, the characterization of the mode of inhibition of three promising inhibitors of the previously-validated drug target P. falciparum thioredoxin reductase (PfTrxR) is reported in this thesis. The enzyme is a homodimeric flavoenzyme which reduces thioredoxin (Trx) via a C-terminally located CysXXXXCys pair. In this respect PfTrxR differs significantly from its human counterpart which bears a Cys-Sec redox pair at the same position. PfTrxR is essentially involved in antioxidant defence and redox regulation of the parasite and has been validated as a drug target. The inhibitors, 4-nitro-2,1,3-benzothiadiazole (IC50 on PfTrxR = 2 µM), 6,7-nitroquinoxaline (IC50 on PfTrxR = 2 µM), and bis-(2,4- dinitrophenyl)sulfide (IC50 on PfTrxR = 0.5 µM), showed uncompetitive inhibition with respect to both substrates, NADPH and thioredoxin. All three inhibitors were active in the lower micromolar range on the chloroquine resistant P. falciparum strain K1. 4-Nitro- 2,1,3-benzothiadiazole was antagonistic with known antimalarials suggesting that the inhibitor uses similar routes of uptake and/or acts on related targets or biochemical pathways (Andricopulo et al., 2005; Andricopulo et al., submitted). Lastly and most importantly, the renaissance of interest in the ancient antimalarial drug methylene blue (MB) led to the identification of a potential artemisinin-based combination therapy (ACT). A strong synergistic action of MB and artemisinin might be capable of fighting resistant P. falciparum parasites in the field. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant P. falciparum strains with IC50 values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high performance liquid chromatography / tandem mass spectrometry on different cell culture compartments, MB accumulates in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine MB showed additive effects. In contrast, synergistic effects of MB with artemisinin, artesunate, and artemether were observed for all tested parasite strains. Artemisinin/MB concentration combination ratios of 3:1 were found to be advantageous demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. In vitro data reported here indicate that combinations of MB with artemisinin (derivatives) might be a promising option for treating drug resistant malaria. Resistance development under this drug combination is unlikely to occur (Akoachere et al., in press). Taken together, the results support the feasibility of the rational development of new potential antimalarial drugs. In combination with existing and other promising new malarial-control measures, new antimalarial drugs could greatly contribute to reducing the intolerable global burden of this disease. Malaria ist eine parasitäre Infektionskrankheit, die von Protozoen der Gattung Plasmodium hervorgerufen wird. Pro Jahr gibt es über 500 Millionen Krankheitsfälle/Neuinfektionen mit 2-3 Millionen Todesfällen. Ein wichtiger Punkt in der Pathogenese der Malaria ist die Ausbildung persistierender Infektionen. Antigenetische Variation ermöglicht es dem Parasiten, das menschliche Immunsystem zu umgehen. Weiterhin sind Plasmodien in der Lage, auf die eingesetzten Malariamittel mit rascher Resistenzentwicklung zu reagieren. Deshalb kommen Neu- und Weiterentwicklung von Medikamenten in der Bekämpfung der Malaria neben Impfstoffentwicklung und Insektiziden Massnahmen gegen den Vektor eine zentrale Rolle zu. Eine Ausnahme in der zunehmenden Resistenzproblematik bildet Artemisinin, welches eine andere chemische Zusammensetzung und einen anderen Wirkmechanismus als andere gegenwärtige Malariamittel aufweist. In Rahmen dieser Doktorarbeit wurden drei neue Arzneimittelentwicklungs-Strategien gegen den gefährlichsten human Malariaereger, P. falciparum, verfolgt. Dies erfolgte anhand von rationaler Medikamententwicklung bzw. durch eine Neubewertung ehemaliger Malariamittel mit dem Ziel, mögliche neue Wirkmechanismen aufzuzeigen und Resistenzentwicklung bei Artemisinin zu verhindern. Der erste dieser verschiedenen Angriffspunkte war die Charakterisierung neuer Charakterisierung eines Glutathion-abhängigen Glyoxalase (Glo) Systems im Vergleich zum isofunktionellen humanen System. Dieses System hat eine zentrale Rolle im Entgiftungsstoffwechsel der Parasiten und besteht aus einer cytosolischen GloI (cGloI), einem Glo-I ähnlichen Protein (GILP), zwei GloII (cytosolische GloII (cGloII) sowie tGloII mit einer vorangestellten Targeting-Sequenz). Hier werden kinetische und strukturelle Ähnlichkeiten im humanen und Plasmodien-System beschrieben und im Sinne einer Überprüfung als möglicher Arzneimittel-Angriffsort Verschiedenheiten aufgezeigt Verbindungen wurden als Inhibitoren der Glyoxalasen an P. falciparum getestet, sie waren im niederen nanomolaren Bereich aktiv. Somit können diese Verbindungen als Leitsubstanzen für die Entwicklung selektiver Inhibitoren des P. falciparum-Glyoxalase Systems dienen (Akoachere et al., 2005). Ein zweiter zentraler Punkt dieser Doktorarbeit ist die Charakterisierung des Wirkmechanismus von drei vielversprechenden Inhibitoren der bereits als Arzneimittel- Zielmolekül validierten P. falciparum Thioredoxinreduktase (PfTrxR). Es handelt sich um ein homodimeres Flavoenzym, welches Thioredoxin mit Hilfe eines C-terminalen CysXXXXCys-Motives reduziert. Hierbei unterscheidet es sich vom humanen Enzym, welches an der gleichen Position ein Cys-Sec Redoxpaar beinhaltet. PfTrxR ist essentiell Abwehrmechanismen Redoxhomöostase Plasmodienstoffwechsel. Die Inhibitoren 4-Nitro-2,1,3-Benzothiadiazol (IC50 für PfTrxR = 2 µM), 6,7-Nitroquinoxalin (IC50 = 2 µM) und Bis-2,4-Dinitrophenyl)sulfid (IC50 = 0,5 µM) zeigen eine unkompetitive Hemmung für die beiden Substrate NADPH und Thioredoxin. Alle drei Inhibitoren sind aktiv im niederen mikromolaren Bereich bei dem choroquinresistenten P. falciparum Stamm K1. 4-Nitro-2,1,3-Benzothiadiazol zeigt einen antagonistischen Wirkmechanismus mit anderen bekannten Malariamitteln; dies bedeutet, dass dieser Hemmstoff entweder einen ähnlichen Aufnahmemechanismus besitzt und/oder Stoffwechselwegen (Andricopulo et al., 2005; Andricopulo et al., submitted). Im Sinne einer Neubewertung früherer Arzneimittel gegen Malaria fokussiert meine Doktorarbeit auf Methylenblau (MB) in Bezüg auf eine mögliche Artemisinin-gestützte Kombinationstherapie (ACT: Artemisinin-based combination therapy). Diese Kombination aus zwei Antimalariamitteln ist ein möglicher Weg, Resistenzenentwicklungen bei Plasmodium zu vermeiden. Methylenblau ist aktiv gegen alle Blutstadien von Plasmodium sowohl an chloroquinresistenten Stämmen mit IC50-Werten im niedermolaren Bereich. Hierbei zeigen Ringstadien die höchste Empfindlichkeit. Darüberhinaus akkumuliert MB in verschiedenen Zellkompartimenten, dies konnte mit Hilfe von Hochdurchsatz- Flüssigkeits-Chromatographie bzw. Tandem-Massen-Spektrometrie gezeigt werden. In Arzneimittel-Kombinations-Assays konnte nachgewiesen werden, dass MB antagonistisch zu Chloroquin und anderen Quinolinen wie Piperaquin und Amodiaquin wirkt, während es mit Mefloquin und Quinine einen additiven Effekt zeigt. Im Gegenteil dazu besitzt MB einen synergistischen Effekt mit Artemisinin, Artesunat und Artemether in allen getesteten Plasmodienstämmen. Ein Konzentrationsverhältnis von 3:1 zwischen Artemisinin und MB hat sich als vorteilhaft erwiesen. Dies verdeutlicht, dass geringe Mengen von MB empfohlen werden können, um maximalen therapeutischen Effekt zu erzielen. Diese hier berichteten in vitro-Daten unterstützen die Thesen, dass die Kombination aus Artemisinin (bzw. Artemisininderivaten) und MB eine vielversprechende Möglichkeit in der therapieresistenter Resistenzentwicklung gegen die Arzneimittelkombination unwahrscheinlich (Akoachere et Zusammenfassend kann man sagen, dass diese Resultate die Eignung der rationalen Arzneimittelentwicklung für neue Antimalariamittel unterstreichen. In Kombination mit existierenden Arzneimitteln und zusammen mit anderen Kontrollmechanismen können neue Antimalariamittel dazu beitragen, die intolerierbare, weltweite Bedrohung durch Malaria zu verringern.


MANUAL PRÁCTICO DE RIEGO Autores: María José Moñino Espino*, Alberto Samperio Sainz-Aja, Antonio Vivas Cacho, Fernando Blanco Cipollone, María del Henar Prieto Losada. Centro de Investigaciones Científicas y Tecnológicas de Extremadura. CICYTEX. Instituto de Investigación Agraria Finca "La Orden-Valdesequera" Departamento de Hortofruticultura. Grupo de riego y nutrició[email protected]

(microsoft word - ley general de la administracion p 332blica f.doc )

LEY GENERAL DE LA ADMINISTRACION PÚBLICA1 DECRETO NÚMERO 146-862 EL CONGRESO NACIONAL, CONSIDERANDO: Que el creciente desarrollo de la actividad social y económica en nuestro país, ha impuesto condiciones a la actividad estatal, que no conviene desatender. CONSIDERANDO: Que el Gobierno de la República, se ha empeñado en la ejecución de los planes nacionales de desarrollo para elevar el nivel de vida a los habitantes y asegurarles su bienestar económico y social.