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1
Maheshwari, Sweta. "Caractérisation biochimique et cellulaire des enzymes clés du métabolisme des phospholipides chez Plasmodium falciparum." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20004.
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Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutiquePhospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach
2
Higham, Christopher W. "A study of lactate dehydrogenase from Plasmodium falciparum." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299529.
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Shoemark, Deborah Karen. "The kinetic characterization of the lactate dehydrogenase enzyme from Plasmodium falciparum." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326677.
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Birkholtz, Lyn-Marie. "Functional and structural characterization of the unique bifunctional enzyme complex involved in regulation of polyamine metabolism in Plasmodium falciparum." Pretoria : [s.n.], 2005. http://upetd.up.ac.za/thesis/available/etd-06302005-120320/.
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5
Birkholtz, Lyn-Marie. "Functional and structural charaterization of the unique bifunctional enzyme complex involved in regulation of polyamine metabolism in Plasmodium falciparum." Thesis, University of Pretoria, 2001. http://hdl.handle.net/2263/25944.
Full textAbstract:
Malaria remains one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite.Thesis (PhD (Biochemistry))--University of Pretoria, 2002.
Biochemistry
unrestricted
6
Turgut, Dilek. "Overproduction of the active lactate dehydrogenase from Plasmodium falciparum opens a route to obtain new antimalarials." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389088.
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Ko, Reamonn, and 高耀駿. "X-ray crystallographic studies of Plasmodium falciparum adenylate kinases." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208020.
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Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010. The most fatal strain of malarial parasite, Plasmodium falciparum is found to contain 3 Adenylate Kinases (PfAK1, PfAK2 and PfGAK). Adenylate Kinases are important enzymes that essentially catalyze and regulate energy metabolism processes. PfAK1 and PfAK2 catalyze the reversible MG2+ reaction ATP + AMP ←→ 2ADP whereas, the PfGAK catalyzes the Mg2+ dependent reaction GTP+AMP ←→ ADP+GDP. Of all malarial strains, only the Plasmodium falciparum Adenylate Kinase 2 (PfAK2) was found to contain a N-myristoylation sequence and subsequently formed a stable heterodimer with Plasmodium falciparum N-myristoyl transferase (PfNMT). The myristoylation of PfAK2 by PfNMT is believed to help transport PfAK2 to the parasitophorous vacuole membrane (PVM) so that the enzyme can perform its essential functions. With these enzymes being key components in the parasite’s survival, the structural study of these enzymes would provide a lot of insight into targeting these proteins for drug design that would effectively kill the parasite without affecting the human host. In this study, PfAK1 was able to be expressed, purified and crystallized with a dataset collected at 4.3Å. PfGAK was expressed and purified. A GTP analogue called GP5A was used to soak the purified PfGAKand the PfGAK bound to GP5A was crystallized and diffracted. Moreover, PfAK2 and PfNMT was successfully expressed and co-purified. The purified PfAK2-PfNMT heterodimer are undergoing crystal screening for possible crystallization conditions.published_or_final_version
Physiology
Master
Master of Philosophy
8
Yao, Jia. "Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1020894.
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Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
9
Khairallah, Afrah. "The identification of natural inhibitory compounds against the plasmodium GTP Cyclohydrolase I (GCH1) enzyme." Thesis, Rhodes University, 2019. http://hdl.handle.net/10962/72284.
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Malaria is a disease caused by protozoan parasites that invade red blood cells causing an infection. Malaria remains a global health problem. The genus Plasmodium infects about a quarter of a billion people annually, resulting in over a million death cases. This can severely affect the public health and socioeconomic development especially in countries with limited resources. Malaria is transmitted by the female Anopheles mosquito. Five species within the Plasmodium genus are known to cause infection in humans; namely Plasmodium falciparum, Plasmodium Ovale, Plasmodium knowlesi, Plasmodium vivax and Plasmodium malariae. The increased resistance by the parasite to the majority of available anti-malarial drugs has raised a great challenge in anti-malarial drug discovery. With the problem of drug resistance on the rise, the need to develop new anti-malarial treatment strategies and identification of alternative metabolic targets for the treatment of malaria is crucial. This study is focused on the Guanosine triphosphate CycloHydrolase I (GCH1) enzyme as a potential drug target. GCH1 is important for the survival of malaria parasites as shown by failed attempts to generate knockout lines in plasmodium falciparum. In this study, sequence and evolutionary analysis were carried out in both the human host and parasite GCH1 enzyme. Accurate 3D models of the parasite GCH1 were built and validated. The resulting models were used for high throughput screening against 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). The high throughput screening was done to identify possible binding sites as well as hit compounds with high selectivity and binding affinity towards the parasite enzyme, this is followed by molecular dynamics simulations to identify protein-ligand complexes and analyze their stability. In this study, a total of five SANCDB compounds were identified as potential inhibitors: SANC00317, SANC00335, SANC00368, SANC00106, SANC00103 and SANC00286. It was found that GCH1 protein can be a potential anti-malarial drug target as it showed selective binding with the inhibitor compounds. The identified inhibitors showed good selectivity and lower free energy of binding towards the parasite GCH1. Force field parameters of GCH1 active site metal were derived and validated. The development of these force field parameters was important for accurate MD simulations of the protein active site; which will allow for future investigation of interactions and stability of the GCH1 protein-ligand complexes.
10
Goolab, Shivani. "Optimization of the heterologous expression of folate metabolic enzymes of Plasmodium falciparum." Diss., University of Pretoria, 2010. http://hdl.handle.net/2263/23647.
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Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements.Dissertation (MSc)--University of Pretoria, 2011.
Biochemistry
unrestricted
11
Contet, Alicia. "Caractérisation biochimique et biophysique des deux cytidylyltransférases de Plasmodium falciparum, enzymes clés du métabolisme des phospholipides." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS085.
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Le paludisme est causé par l'infection et la destruction des érythrocytes par les parasites protozoaires appartenant au genre Plasmodium. Au cours de son développement dans l'érythrocyte,Plasmodium falciparum requiert la biosynthèse massive de membranes dont les principaux constituants lipidiques sont des phospholipides. La phosphatidylcholine (PC) et la phosphatidyléthanolamine (PE) représentent à elles deux environ 80 % des lipides membranaires et l'inhibition de leur biosynthèse est létale pour le parasite. La PC et la PE sont synthétisées par le parasite, principalement via les voies de novo dépendantes de la CDP-choline et de la CDP-éthanolamine (ou voies de Kennedy) en utilisant respectivement la choline et l'éthanolamine comme précurseurs. Ces travaux de thèse se focalisent sur les deux enzymes CTP:phosphocholine etCTP:phosphoéthanolamine cytidylyltransférase (PfCCT et PfECT, respectivement), catalysant les étapes limitantes des voies de Kennedy. Chez Plasmodium, les CCT et ECT possèdent deux domaines cytidylyltransférases (CT) portant l'activité catalytique, séparés par une longue région de liaison. Pour la CCT, cette duplication est retrouvée seulement chez trois organismes, tous faisant partie du phylumdes Apicomplexes : Babesia, Theileria et Plasmodium, alors que la présence de deux domaines CT estune caractéristique retrouvée chez toutes les ECT étudiées à ce jour. La première partie de ce travail de thèse concerne la caractérisation biochimique et l'inhibition la PfCCT Nous avons montré que les deux domaines CT de la PfCCT sont actifs à l'inverse de la PfECT pour laquelle seul le domaine CTN-terminal est catalytiquement actif. A la suite d'un criblage virtuel basé sur la structure de l'enzyme,nous avons identifié un composé princeps capable d'inhiber l'activité de la PfCCT in vitro, la synthèse de PC et la croissance parasitaire. Ce premier composé actif (haut µM) représente une base pour l'optimisation future de nouveaux composés plus efficaces. Dans la deuxième partie de cette thèse,nous avons déterminé le mécanisme catalytique, la spécificité de liaison des ligands et l'organisation structurale de la PfECT grâce à la combinaison d'approches biochimiques et biophysiques. L'ensemble des résultats présentés dans ce manuscrit apportent un éclairage important concernant le fonctionnement de ces deux cibles potentielles et constituent des étapes essentielles à l'élaboration d'une approche thérapeutiqueMalaria is caused by the infection and destruction of red blood cells by protozoan parasitesbelonging to the genus Plasmodium. During its intra-erythrocytic development, Plasmodiumfalciparum requires massive biosynthesis of membranes which are mainly composed of phospholipids.Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together represent about 80% of thetotal membrane lipids and inhibition of their biosynthesis leads to parasite death. PC and PE aresynthesized by the parasite's machinery mainly through the de novo CDP-choline and CDPethanolamine(Kennedy) pathways using respectively choline and ethanolamine as precursors. Thisstudy focuses on the rate limiting steps of these pathways catalyzed by CTP:phosphocholine andCTP:phosphoethanolamine cytidylytransferases (PfCCT and PfECT, respectively). In Plasmodiumspecies, both CCT and ECT contain two catalytic cores (CT domains) separated by a long linker.Interestingly, for CCT this feature is found only in three organisms, all from the phylum ofApicomplexa: Babesia, Theileria and Plasmodium, whereas the presence of two CT domains is ageneral feature in all ECTs known so far. The first part of this work consists in the biochemicalcharacterization of PfCCT and the investigation of its druggability. We showed that both PfCCT CTdomains are active and display similar kinetic parameters while only the N-terminal CT domain wasactive in PfECT. Subsequent to an in silico structure-based screening of compounds libraries, weidentified a PfCCT inhibitor able to inhibit PC synthesis as well as P. falciparum growth in vitro in thehigh µM range. This compound represents a first step toward the optimization of future more potentcompounds. In the second part of this study, we investigated the catalytic mechanism of PfECT anddeciphered its interactions with its ligands using biochemical, biophysical and structural approaches.Collectively, these results bring new insights into the biochemical and structural properties of thesetwo keys enzymes of the phospholipid metabolism in P. falciparum and pave the way for their futuredevelopment as potential drug target
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Goble, Jessica Leigh. "The druggable antimalarial target 1-deoxy-D-xylulose-5-phosphate reductoisomerase: purfication, kinetic characterization and inhibition studies." Thesis, Rhodes University, 2011. http://hdl.handle.net/10962/d1004008.
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Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.
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Tran, Thanh Nguyen. "Plasmodium Falciparum Histone Deacetylases as Novel Antimalarial Drug Targets." Thesis, Griffith University, 2010. http://hdl.handle.net/10072/367456.
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Histone deacetylases (HDACs) are recognised as potential drug targets for many diseases including
cancer, inflammatory diseases and some parasitic diseases including malaria. In eukaryotic cells,
these enzymes play an important role in transcriptional regulation through modification of
chromatin structure. Inhibitors of mammalian HDAC enzymes including trichostain A and apicidin
are active against P. falciparum parasites, however these compounds are not selective for malaria
parasites versus normal cell lines. The aims of this study were to examine the antimalarial potential
of new hydroxamate-based HDAC inhibitors and to investigate a P. falciparum HDAC, PfHDAC1,
as a potential new antimalarial drug target.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Science
Griffith Health
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Njuguna, Joyce Njoki. "Structural analysis of prodomain inhibition of cysteine proteases in plasmodium species." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004081.
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Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
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Abd, Majid Roslaini. "Molecular and biochemical pharmacology of mitochondrial enzymes in the malaria parasite Plasmodium falciparum." Thesis, University of Liverpool, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574668.
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The mitochondria of malaria have big potential to be explored as a drug targeting site. This is due to the differences in the composition of the Plasmodium respiratory complex compared with the human host. The Plasmodium respiratory chain consists of 2 unique dehydrogenases PfNDH2 and MQO which are only encoded in prokaryotic cells. Apart from these two, thy Plasmodium bc1 complex has low amino acid similarity when compared to the bc1 cQmplex of other eukaryotes. Currently the only approved antimalarial drug targeting Plasmodium mitochondria is atovaquone. This drug is used in combination with proguanil (Malarone™) and is widely used for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc] complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential and subsequent parasite death. However, atovaquone resistance developed very soon after its implementation as malaria chemotherapy. Previous studies have established that the resistance of the parasite is the result of mutations in the bc1 Q0. To date we are the first group to elucidate the genotype and biochemical characterisation of the atovaquone resistant P. Jalciparum TM90C2B. The GeXP multiplex quantitative PCR (qPCR) revealed that a number of genes encoding energy metabolism proteins and genes associated with redox control are upregulated in the resistant parasite compared to the atovaquone sensitive strain 3 D7. This has been supported by automated sequencing results which indicate the presence of a single point mutation in the DNA sequence of TM90C2B cytochrome b which resulted in the substitution of tyrosine by serine at position 268. The drug sensitivity assays and kinetic studies were conducted in order to understand the phenotypic consequences of this mutation. As expected, TM90C2B strain was resistant to almost all electron transport chain inhibitors. The enzymological characterisation of TM90-C2B bc] complex (steady-state decylubiquinol:cytochrome oxidoreductase assay) showed that enzyme turnover was approximately 50% of the atovaquone-sensitive strain with a threefold increase in Krn for decylubiquinol (3D7 : Vrnax = 97.4 ± 5.1 nmol cyt c reduced/min/mg protein, Krn = 5.5± 1.1 mM dQH2, ICSD 6 nM, K; = 0.6 nM; TM90C2B : Vrnax = 60.2 ± 3.2 nmol cyt C reduced/min/mg protein, Krn = 18.5± 2.6 mM dQH2, leSD 600 nM, K; = 162 nM). The other mitochondrial respiratory complexes' specific activities were also measured using samples prepared from Percol® fractionation. Apart from Complex II, we also managed to demonstrate the specific activities of the other complexes'. The PfNDH2 and bCI complex proteins ofTM90C2B strain had 50% less activity than the 3D7 strain. This was supported by data from western blot analysis showing a decrease in PfNDH2 and ISP proteins content of the TM90C2B compared to the 3D7 strain. In addition to this study, we also characterised the Plasmodium Jalciparum mitochondrial malate quinone oxidoreductase (MQO). MQO is involved in the electron transport chain, oxidising malate to oxaloacetate in the oxidative arm of Plasmodium tricarboxylic acid cycle (TCA cycle). This enzyme contains a flavin cofactor which donates electrons to cytochrome bCI complexvia reduction ofubiquinone to ubiquinoL The bioinformatic analysis data indicates that PfMQO is a membrane-bound mitochondrial protein with only one transmembrane domain. The PfMQO gene was successfully amplified from genomic DNA of 3D7 P. falciparum and cloned into 2 expression vectors pET-15b and pUCI9. The presence and orientation of the gene in the respective vector were confirmed by restriction enzyme analysis and automated sequencing. The putative PfMQO gene (1566 bp) was successfully amplified and predicted to produce an approximately 60-kDa protein. Only when the PfMQO gene was cloned into pET15b was overexpression of the recombinant protein achieved. The recombinant PfMQO was purifiable under denaturing conditions due to its insolubility and formation of inclusion bodies. This protein is inactive and appears to be improperly folded thus producing the inconsistent results of the kinetic analyses. In conclusion, our study provides new insights into the understanding of the moleCular and biochemical regulation of atovaquone resistant parasites through a mutation in the bc1 complex. The partial characterisation of PfMQO is a starting point for the development of a new drug target in Plasmodium mitochondria by taking into account the uniqueness of this enzyme which is not found in the human host.
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Southworth, Paul. "Quantitative proteomics of the human malaria parasite, Plasmodium falciparum, applied to folate biosynthetic enzymes." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/quantitative-proteomics-of-the-human-malaria-parasite-plasmodium-falciparum-applied-to-folate-biosynthetic-enzymes(3ba6c57f-3f37-443d-92d9-b255722e3f69).html.
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Human malaria caused by Plasmodium falciparum is a major global burden killing between 700,000 and 2.7 million people every year. Africa bears the greatest portion of this burden, with over three quarters of deaths occurring in African children, accounting for 18% of all child deaths in sub-Saharan Africa. Synthesis of tetrahydrofolate through the folate biosynthetic pathway is vital for the survival of P. falciparum parasites and is lacking in the human host. As such, enzymes of this pathway have long presented attractive targets for drug therapy and although increasingly being compromised by resistance, anti-folates such as pyrimethamine and sulfadoxine are still very valuable drugs in many malaria-endemic regions.In this project, further investigation of the enzymes of the folate biosynthetic pathway has been attempted by developing protocols to quantify these proteins and others through proteomic techniques. Two quantification techniques were pursued. The first was quantification using whole, heterologously expressed, stable-isotope labelled forms of P. falciparum proteins for use as heavy standards in mass spectrometry. Great difficulty was experienced in the effort to express and purify P. falciparum enzymes in E. coli expression systems, with only one enzyme successfully expressed and purified in a 13C-labelled form. This one protein was taken forward into quantification experiments. The second quantification technique used a stable-isotope labelled ‘QConcat’ protein, consisting of a number of peptides from 12 P. falciparum proteins of interest, as a heavy standard in mass spectrometry. This was successfully expressed and purified in a 13C-labelled form from an artificial gene using an E. coli expression system. This too was taken forward into quantification experiments.Quantification experiments using the QConcat-based quantification technique were successfully performed on whole P. falciparum extract. Among the proteins quantified were SHMT and DHFR, two proteins of great interest from the folate biosynthetic pathway. Consistent with results from different expression analysis techniques in the literature, the folate enzymes were found to be of lower abundance than housekeeping enzymes and SHMT was found to be more abundant than DHFR.For deep quantitative analysis of the P. falciparum proteome, it was found that fractionation was necessary. Fractionation in this project was performed using a ZOOM™ IEF fractionator (Invitrogen), an OFFGEL™ IEF fractionator (Agilent) and 1D SDS-PAGE. It was found that by using these fractionation techniques, more proteins could be identified within the P. falciparum proteome, with all but one of the enzymes of the folate biosynthetic pathway being identified. Significant advances in the sensitivity of mass spectrometers during this project have also greatly facilitated the investigation of the proteome. In some cases, this meant that proteins which were only previously accessible by prefractionation of the proteome could be seen in whole P. falciparum extract. Unfortunately, QConcat-based quantification using both fractionation and sensitive mass spectrometry could not be successfully achieved in the time available. However, the promising results obtained suggest that, after careful optimisation, such an approach will be valuable.
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Engel, Jessica Alexandra. "Investigating Plasmodium falciparum Histone Deacetylase 1 Complex Proteins." Thesis, Griffith University, 2017. http://hdl.handle.net/10072/367801.
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Malaria causes substantial morbidity and mortality worldwide. Although there has been a considerable decline in global malaria incidence and mortality rates since 2000, it is estimated that more than 400,000 deaths occurred in 2015 as a result of this parasitic disease. The lack of a broadly effective licensed vaccine and the threat of malaria parasite resistance to current drugs means there is an urgent need for the development of new therapies with novel parasite targets. With a renewed call for global malaria eradication, novel therapeutic strategies are crucial to continue progress achieved over the last decade in combating malaria and to achieving a malaria-free world.
Targeting epigenetic mechanisms within Plasmodium parasites represents a promising therapeutic approach for malaria. Histone deacetylase (HDAC) enzymes, the focus of this thesis, act in conjunction with histone acetyltransferases (HATs) to reversibly acetylate histone and non-histone proteins. Some HDACs, in particular class I and II HDACs, are already validated drug targets for cancer therapy and are showing promise as antimalarial drug targets. However, besides their classical role in regulating gene expression, knowledge of the roles that HDACs play in the Plasmodium parasites, is limited. Like higher eukaryotic HDACs, P. falciparum HDACs (PfHDACs) are believed to localise in multi-protein complexes with accessory proteins and to regulate lysine acetylation of both histones and non-histone proteins.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Wang, Flora Yinglai-Hua. "Purification and Characterization of Native and Recombinant Dipeptidyl Aminopeptidase 1 of Plasmodium falciparum." Thesis, Virginia Tech, 2008. http://hdl.handle.net/10919/42714.
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Plasmodium falciparum dipeptidyl aminopeptidase 1 (DPAP1) contributes to the degradation of hemoglobin by releasing dipeptides from globin oligopeptides in the food vacuole. The lack of success at DPAP1 gene disruption suggests that this exopeptidase is important for efficient growth during the erythrocytic asexual stage. DPAP1 is therefore an attractive target for the development of anti-malarial drugs that block the catabolism of hemoglobin. To guide the design of selective, potent DPAP1 inhibitors, it is necessary to characterize the substrate specificity of this enzyme along with its human homolog cathepsin C. Although native purification of DPAP1 is possible, the amount of purified enzyme obtained is insufficient for extensive biochemical characterization. To overcome this obstacle, a strategy was developed for the recombinant expression of soluble DPAP1 in the bacterium Escherichia coli and for its activation in vitro. The production of active recombinant DPAP1 presents three challenges: 1) expression of the protein in soluble form, 2) generation of the native N-terminus, and 3) cleavage of the pro-domain. Soluble expression of DPAP1 was achieved by fusing it to the C-terminus of maltose-binding protein (MBP). A linker sequence encoding a tobacco etch virus protease (TEVp) cleavage site was introduced between MBP and DPAP1 such that TEVp cleavage would generate the presumed native N-terminus of DPAP1. Incubation of the MBP-DPAP1 fusion with TEVp resulted in the release of free DPAP1which hydrolyzed the fluorogenic substrate proyly-arginyl-7-amido-4 methyl coumarin (Pro-Arg-AMC). Various proteases were tested for the ability to excise the pro-region. Treatment with both trypsin and papain removed the pro-region and increased DPAP1 activity two to three fold. When assayed with Pro-Arg-AMC, trypsin-treated DPAP1 had kinetic properties similar to native enzyme whereas papain-treated DPAP1 deviated from Michaelis-Menten kinetics. Using a combinational dipeptidyl substrate library, the substrate specificities of native and recombinant (trypsin-activated) DPAP1, as well as of human cathepsin C were profiled. We find that both DPAP1 and human cathepsin C accept a wide spectrum of amino acid side chains at the substrate P1 and P2 positions. Interestingly, several P2 residues show high selectivity for DPAP1 or cathepsin C. The collected data point to the feasibility of designing inhibitors that are specific for DPAP1 over cathepsin C.Master of Science in Life Sciences
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Gabriel, Heloisa Berti. "Caracterização funcional de farnesil difosfato sintase/geranilgeranil difosfato sintase (FPPS/GGPPS) e 1,4-dihidroxi-2-naftoato preniltransferase (MenA) envolvidas respectivamente na via de isoprenóides e da vitamina K em Plasmodium falciparum." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-22022016-153037/.
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A malária é uma das principais e a mais disseminada das parasitoses humanas. A falta de uma vacina eficaz e o problema da resistência aos fármacos tem contribuído para o adiamento da solução do controle desta infecção. A busca de novos alvos biológicos tem se concentrado, em parte, na compreensão de vias metabólicas. Em P. falciparum, identificamos a biossíntese das duas formas da vitamina K (filoquinona e menaquinona). Na via MEP foram caracterizadas duas importantes enzimas bifuncionais, a farnesil difosfato sintase/geranilgeranil difosfato sintase (FPPS/GGPPS) capaz de formar farnesil difosfato e geranilgeranil difosfato e octaprenil pirofosfato sintase/fitoeno sintase (OPP/PSY) responsável pela biossíntese da cadeia isoprênica que se liga ao anel da via de ubiquinona, como também forma o primeiro caroteno na via de carotenóides. Este projeto tem como objetivo caracterizar o gene MenA da biossíntese de MQ, determinar a localização de FPPS/GGPPS em P. falciparum e investigar a importância de OPP/PSY e de FPPS/GGPPS no ciclo intraeritrocítico de P. falciparum.Malaria is one of the main widespread human parasites. The lack of an effective vaccine and the problem of drug resistance haves contributed to the delay of the control solution of this infection. The search for new biological targets has focused in part on the understanding of metabolic pathways. In P. falciparum, identified the biosynthesis of the two forms of vitamin K (phylloquinone and menaquinone). In the MEP pathway were characterized two important bifunctional enzyme, farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) able to form farnesyl diphosphate and geranylgeranyl diphosphate and octaprenyl pyrophosphate synthase/phytoene synthase (OPP/PSY) responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, but also forms the first carotene in the carotenoid pathway. This project aims to characterize the MenA gene from the MQ biosynthesis, determine the localization of FPPS/GGPPS and investigate the importance of OPP/PSY and FPPS/GGPPS in intra-erythrocytic cycle of P. falciparum.
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Pye, Matthew James. "Inhibiting Plasmodium falciparum IspD, a MEP pathway enzyme, as a novel target for the development of antimalarial chemotherapeutics." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3020589/.
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Reeksting, S. B. (Shaun Bernard). "Targeted inhibition of the Plasmodium falciparum Vitamin B6 producing enzyme Pdx1 and the biochemical and functional consequences thereof." Thesis, University of Pretoria, 2013. http://hdl.handle.net/2263/32965.
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Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials.Thesis (PhD)--University of Pretoria, 2013.
gm2013
Biochemistry
Unrestricted
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NETO, Zoraima Naymbi da Silva. "Biological characterization of de-ubiquitylating enzymes (UBPs/UCHs) in Plasmodium spp as potential drug targets." Doctoral thesis, Instituto de Higiene e Medicina Tropical, 2014. http://hdl.handle.net/10362/19274.
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A malária ainda constitui um grande problema de saúde pública e a resistência aos antimaláricos ameaça todos os esforços efectuados com vista ao combate e controle desta doença. Existe uma grande necessidade de se identificar novos compostos de preferência que actuem em novos alvos terapêuticos. A via da ubiquitinação/proteosoma já foi identificada como um alvo terapêutico interessante. Mutações nas enzimas de des-ubiquitilação (DUBs) que catalizam a remoção da ubiquitina estão associadas ao desenvolvimento de doenças infecciosas e não infecciosas.
Neste projecto quatro DUBs foram identificadas no genoma do parasita Plasmodium falciparum e foram caracterizadas. A expressão dos genes que codificam estas enzimas ao longo do ciclo de vida do parasita na presença e ausência de fármaco foi efectuada por RT-PCR.Anticorpos policlonais obtidos a partir de ratinhos foram utilizados para a deteção da abundancia das proteínas ao longo do ciclo de vida do parasita. Utilizou-se ainda a tecnica de transfeção com o objectivo de criar uma linha knockout para determinar se estas proteínas são essências para o parasita. Proteínas recombinantes foram expressas em células de E.coli e actividade enzimática das mesmas foi testada usando um substrato específico para as DUBs. O inibidor das DUBs com actividade antimalarica, curcumina foi usado quer in vitro para testar a sua actividade sobre as proteínas recombinantes, mas também in vivo no modelo de malaria roedora de Plasmodium chabaudi em associação com cloroquina e artemisinina.Um ensiao de proteomica foi também usado para ver que proteínas estão alteradas em resposta ao tratamento com curcumina.
Os resultados demonstram que em P. falciparum os genes pfuch-l1, pfuch-l3, pfuch-l54 e pfubp-8 são diferencialmente expressos ao longo do ciclo de vida do parasita e as respectivas proteínas são mais abundantes no estadio de trofozoito e esquizonte. O tratamento dos parasitas com artemisinina, cloroquina, curcumina induziu um aumento temporário na expressão dos genes seguido de um declínio. Não foi possível obter uma linha parasitária knockout pfuch-l1 e pfuch-l3 viável. As proteínas recombinantes foram expressas com sucesso em células de E. coli excepto a Pfuch-l54. As Pfuch-l1, Pfuch-l3, Pfubp-8 demonstraram actividade enzimática e interagiram com o susbstrato Ub-AMC. Os IC50 da curcumina nas proteínas recombinantes foram: Pfuch-l1 15μM, Pfuch-l3 25.4μM, Pfubp-8 10μM e para a proteina recombinante humana USP2, 5μM. A Curcumina quando testada nas células HepG2 apresenta alguma toxicidade in vitro, mas não apresenta uma alta toxicicidade em ratinhos e quando utilizada em associação com a cloroquina apresenta um efeito de sinergismo.Enquanto a associação da curcumina com artemisinina o resultado é antagónico.Os ensaios de proteomica em culturas de P. falciparum tratadas com curcumina revelaram 10 proteinas que se encontraram alteradas em resposta ao tratamento. Estas proteínas estão envolvidas no metabolismo do sulfato, tradução e degradação de proteínas, ciclo celular e organização celular.
Em conclusão, este trabalho demonstra que estas enzimas são potenciais alvos terapeuticos, mas será necessário mais estudos moleculares, bioquímicos e farmacológicos para aumentar a selectividade dos inibidores das DUBs para as enzimas do parasita e minimizar os danos nas proteínas do hospedeiro humano.Malaria continues to be a major public healthconcern. Drug resistance continues to threaten all efforts made to control the disease. Hence there is a race to identify new antimalarial drugs that act on newer targets, in order to minimize the spread of drug resistance. The ubiquitin/proteasome pathway has been idientified as a potential drug target. Mutations in de-ubiquitylating enzymes (DUBs),which catalyze the removal of ubiquitin,havebeen associated with the developmentof infectiousand non infectious diseases. In this project four DUBs namely pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8were identified in the Plasmodiumfalciparumgenome and were characterized. Theexpression profile of genes encoding DUBsthroughout the parasite ́s life cycle with and without drug treatment wascarried out by RT-PCR.Polyclonal antibodies raised in mice were used to detect protein abundance in different stages of the parasite ́s life cycle. An attempt was made to produce a DUB knockoutlineand determine whether they are essential for the parasite. Recombinant proteins were expressed in E.colicells and their de-ubiquitylating activity was tested usinga specific substrate for DUBs.The activity of curcumin (a Dub inhibitor)was evalutedinvitroon the recombinant proteinsand its antimalarialactivity was testedin association with chloroquine and artemisininin anin vivorodent malaria model,Plasmodium chabaudi. A proteomics approach was also used to determine what proteins were deregulated in response to curcumin treatment.The results show that P.falciparumgenes pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8are differentially expressed throughout the parasite ́s life cycle and those proteins are more abundant at the trophozoite and schizont stages of the parasite.Treatment of parasites with artemisinin, chloroquine, and curcumin induced a transientincrease in the expression of those genes,followed by a steadydecrease in the gene expressionpattern.No viable pfuch-l1and pfuch-l3gene knockout lines were obtained.Recombinant proteins were successfully expressed in E.colicellswith the exception of Pfuch-l54.Pfuch-l1, Pfuch-l3, Pfubp-8demonstrated de-ubiquitylating activity by cleaving the substrate Ub-AMC. In vitroIC50 of curcumin towards recombinant Pfuch-l1was 15μM, for recombinant Pfuch-l3was 25.4μM and forPfubp-8was 10μM and for human USP2 was 5μM. Curcumin displayed some toxicity to the HepG2 cell lines, but the in vivoantimalarial activityassays in the rodent model of malaria Plasmodium chabaudishowedthat curcumin is non toxic to miceand the association of curcumin with chloroquine displayedsynergism whereas theassociation of curcumin with artemisinin showed antagonism. The proteomics assay performed in P.falciparumcultures treated with curcuminrevealed10deregulatedproteins.The proteins identified were involved in sulfur metabolism, protein translation and degradation, cell cycleand cellular organization. In conclusion, the presentstudyshowed that P.falciparumDUBs are indeed potential drug targets. However further molecular, biochemicaland phamacological studies will be required in ordertoturn the inhibitors more specific towards the parasite ́s enzymes andminimise damage to the host ́s proteins.
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Mokoena, Fortunate. "Malarial drug targets cysteine proteases as hemoglobinases." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004065.
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Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
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Hari, Har Joshi Srisin Khusmith. "Monoclonal antibody based ELISA for the detection of P. falciparum and P. vivax antigens in Malaria endemic populations in southern Nepal /." Abstract, 2003. http://mulinet3.li.mahidol.ac.th/thesis/2546/46E-Hari-J.pdf.
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Silva, Márcia Ferreira da. "Estudos in vitro de potenciais antimaláricos nos estágios intraeritrocítico de Plasmodium falciparum." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-23042013-122652/.
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Nesta dissertação, identificou-se que as drogas esqualestatina, fosmidomicina, risedronato e nerolidol apresentam atividades sinérgicas e aditivas quando administradas em cultura de P. falciparum. Esses resultados contribuem para a compreensão da biologia do parasita e abrem estudos para possíveis antimaláricos. Identificou-se a especificidade da droga esqualestatina inibidora da enzima fitoeno sintase por meio de marcações metabólicas utilizando precursor radioativo ([3H]GGPP), e análise pela técnica de cromatografia (RP-HPLC). Realizou-se testes de inibição para determinar o valor da IC50 na linhagem pRM2-Fito-HA, a qual encontra-se super expressando enzima fitoeno sintase e encontrou-se um valor IC50 de 5 mM para o isolado 3D7 enquanto que para a linhagem pRM2-Fito-HA foi de 30 mM. Demonstrando assim que a enzima fitoeno sintase é o principal, senão único alvo da esqualestatina em P. falciparum, o que sugere que este composto ou derivado do mesmo são potenciais antimalaricos.In this thesis, we found that the drug squalestatin, fosmidomicina, risedronate, nerolidol have synergistic and additive activity when administered in cultured P. falciparum. These results contribute to the understanding of the biology of the parasite and open studies for potential antimalarials. We identified the specific drug squalestatin inhibiting phytoene synthase enzyme by using metabolic markers radioactive precursor ([3H] GGPP) by the technique of analysis and chromatography (RP-HPLC). Held inhibition tests to determine the IC50 value of the strain in pRM2-Phyto-HA, which is super expressing phytoene synthase enzyme and met an IC50 value of 5 microM to isolate 3D7 whereas for strain pRM2 -Phyto-HA was 30 mM. Thus demonstrating that the enzyme phytoene synthase is the primary, if not sole target of squalestatin in P. falciparum, which suggests that this compound or derivative thereof as potential antimalarials.
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Alberge, Blandine. "Choline et éthanolamine kinases chez Plasmodium falciparum : Caractérisation biochimique et cellulaire des enzymes et de leur activité." Montpellier 1, 2009. http://www.theses.fr/2009MON13517.
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Le développement de Plasmodium falciparum, agent du paludisme, est indissociable d’une production considérable de membranes par le parasite. Les biosynthèses de phosphatidylcholine (PC) et de phosphatidyléthanolamine (PE), principaux constituants de ces membranes, sont essentielles à la croissance du parasite au cours de son développement intraérythrocytaire, phase durant laquelle sont observés les symptômes cliniques de la maladie. Les travaux de cette thèse ont eu pour objectifs la caractérisation des premières enzymes des voies de synthèse de novo de PC et de PE chez P. Falciparum, soit respectivement la choline kinase (PfCK) et l’éthanolamine kinase (PfEK). Les paramètres cinétiques ont été déterminés sur les kinases recombinantes exprimées en système bactérien puis purifiées, ainsi que sur les protéines endogènes provenant d’extraits parasitaires. La PfCK et la PfEK paraissent spécifiques de leur substrat respectif, à savoir la choline ou l’éthanolamine. Nous avons étudié l’inhibition des kinases recombinantes et endogènes, par des composés analogues de substrat et par une nouvelle molécule antipaludique T3/SAR97276A, actuellement en essais cliniques de phase II. Chacune de ces enzymes est inhibée spécifiquement par l’analogue de substrat. D’autre part, T3, bien que conçu comme un analogue de choline, apparait être un inhibiteur compétitif de la PfCK mais également de la PfEK. Nous avons immunisé des souris avec les protéines recombinantes purifiées. Au moyen des séra polyclonaux murins reconnaissant spécifiquement la PfCK ou la PfEK, nous avons montré que ces kinases sont localisées dans le cytoplasme de P. Falciparum et sont exprimées à tous les stades du cycle érythrocytaire de ce parasite, particulièrement au stade mature. Nous avons caractérisé des souches de P. Falciparum transgéniques surexprimant la PfCK ou la PfEK. La sensibilité de ces souches a été mesurée vis-à-vis des analogues de substrat, de T3 et de composés antipaludiques afin d’élucider l’impact, in situ, de ces composés sur la croissance des parasitesThe growth of Plasmodium falciparum, a vector of malaria, within human erythrocytes, is linked to a huge production of membranes. Biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the main phospholipids of malarial membranes, is crucial for the parasite survival. In this thesis, we expose the characterization of the first enzymes of the de novo biosynthesis pathways of PC and PE in P. Falciparum, choline kinase (PfCK) and ethanolamine kinase (PfEK) respectively. Kinetic parameters of both enzymes have been determined, in vitro, on purified recombinant proteins and endogenous ones. PfCK and PfEK are specific for their respective substrates, choline or ethanolamine. We studied the inhibition of both recombinant and endogenous kinases by specific substrate analogs and by a new anti-malarial compound T3/SAR97276A, which is currently in clinical trials. These enzymes were specifically inhibited by the respective substrate analog. In contrast, T3 designed as a choline analog, affected similarly PfCK and PfEK activities. We immunized mice with recombinant pure proteins. With the specific polyclonal murine sera, we localized PfCK and PfEK in the parasitic cytoplasm. These kinases are increasely expressed during the intra-erythrocytic life cycle of P. Falciparum. We also characterized malarial transgenic strains over-expressing PfCK or PfEK, to determine the effect of inhibitory compounds on the intra-erythrocytic growth of P. Falciparum
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GALBIATI, ANDREA. "DESIGN AND SYNTHESIS OF NOVEL ENZYME INHIBITORS AS ANTIPROLIFERATIVE COMPOUNDS WITH ANTIPROTOZOAL AND ANTICANCER ACTIVITY." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/827428.
Full textAbstract:
This dissertation describes the research carried out as part of a PhD program in Chemistry
from the 1st October 2017 until 30th November 2020. The PhD project investigated the
development of inhibitors of enzymes involved in important metabolic pathways, with the
final aim to produce an antiproliferative effect. The present thesis combines the works
performed at the University of Milan and Vrije Universiteit of Amsterdam.
Part A describes the research performed in Amsterdam, NL during my period abroad from
January to September 2019 in the research group of Professor Rob Leurs, at the Division of
Medicinal Chemistry of the Amsterdam Institute for Molecules, Medicines and Systems,
Vrije Universiteit of Amsterdam. In particular, this part outlines the design, synthesis and
pharmacological evaluation of two novel series of potent antitrypanosomal agents,
identified through SAR exploration and scaffold hopping approach starting from cyclic
nucleotide Trypanosoma brucei phosphodiesterase (PDE) inhibitors. PDE enzymes provide
a fine control on several biochemical pathways and have recently been demonstrated to
be essential for parasite proliferation. Their disruption by RNA interference (RNAi)
dramatically increase intracellular cAMP and, consequently, causes complete mortal
trypanosome cell lysis.
Part B describes the research done at the Department of Pharmaceutical Sciences,
University of Milan, under the supervision of Professor Paola Conti, on the design and
synthesis of novel covalent inhibitors targeting the glycolytic enzyme Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). Due to its pivotal role in the glycolysis, GAPDH
represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this
pathway for energy production. In this context, GAPDH inhibition represents a valuable
approach for the development of anticancer and antiparasitic drugs. Due to the presence
of a druggable nucleophilic cysteine residue in the catalytic pocket of the target, I focused
my attention on the development of covalent GAPDH inhibitors, presenting an electrophilic
warhead with a finely tuned reactivity. In particular, Section B2 reportsthe work conducted
on the development of Plasmodium falciparum GAPDH inhibitors and the in vitro
antiplasmodial activity. Section B3 shows the work performed on the design and synthesis
of human GAPDH inhibitors, with in vitro antitumor activity.
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Battistini, Matthew R. "Novel Enzyme Perspectives: Arylalkylamine N-acyltransferases from Bombyx mori & 1-Deoxy- D-Xylulose-5-Phosphate Synthase from Plasmodium falciparum and Plasmodium vivax." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5908.
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This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction.
Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding arylalkylamide by the enzyme arylalkylamine N-acyltransferase (AANAT). In insects, this enzyme is responsible for controlling melanism, the inactivation of biogenic amines, the sclerotization of the insect cuticle, photoperiodism, and the penultimate intermediate in the production of melatonin. We studied a pair of AANAT enzymes from B. mori: Bm-AANAT and Bm-AANAT3. The former was found to catalyze the direct formation of long-chain acylarylalkylamines (only the second enzyme discovered to do such chemistry), while the latter exhibited potent inactivation of several amines through acetylation. We conducted a full kinetic characterization of Bm-AANAT3, including double-reciprocal plots, pH-rate profiling, dead-end inhibition, and the construction of mutants to elucidate catalytically-relevant amino acids. Our hope is that new insights and discoveries on these enzymatic pathways in model organisms will yield novel molecular targets for human health and disease.
We then developed an innovative, microwave-assisted synthesis of a binding-based probe capable of enriching proteins that bind adenine ribose derivatives (AdoRs). We employed this probe in activity-based protein profiling studies to qualitatively assess the AdoR-binding proteome in three bacterial cell lines from the genus Bacillus. This proof of concept experiment demonstrated a unique subset of proteins distinctive to each species, and confirmed the efficacy of the probe tagging and subsequent enrichment. This technology can be used in clinical applications for the detection and identification of cancerous biomarkers.
Finally, we successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these TPP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other TPP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.
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ROSA, M. DE. "Characterization of ferredoxin-NADP+ reductases from pathogenic microorganisms." Doctoral thesis, Università degli Studi di Milano, 2007. http://hdl.handle.net/2434/59919.
Full textAbstract:
Ferredoxin-NADP+ reductase (FNR, EC 1.18.1.2) is a functional class of proteins that catalyzes the transfer of electron equivalents from small iron-sulphur proteins (ferredoxin, Fd) to pyridine dinucleotide, or vice versa. Two different, phylogenetically unrelated, subclasses of FNR exist: the plant-like and the mitochondrial FNRs. FprA is a mycobacterial reductase belonging to the latter class. We discovered that FprA is able to catalyse an unusual reaction, leading to the production of a modified NADP, named NADPO. I characterized its production and I showed that this reaction is a hallmark of the mitochondrial-type of FNR. Moreover, in collaboration with the group run by Prof. Bolognesi, we crystallized and solved the structure of a 7Fe ferredoxin, identified as the most probable substrate of FprA. Plan-type FNR has also been found in some human parasites, including Plasmodium falciparum (the causative agent of the tropical malaria). As FNR does not exist in animals but it is important for the parasites, it has been reported to be good drug target for novel therapies against Apicomplexa. In relation to this finding, using different approaches, I looked for inhibitors of this enzyme by screening a phage-display peptide library and by in silico screening of a compound library using the FNR 3D structure as target. Moreover, I characterized some compounds, reported to be good inhibitors of other FNRs or related enzymes.
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Jordão, Fabiana Morandi. "Caracterização da enzima bifuncional farnesil difosfato/geranilgeranil difosfato sintase e efeito do risedronato nos estágios intraeritrocitários de Plasmodium falciparum." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-23042013-093744/.
Full textAbstract:
O aumento da resistência do parasita da malária a maioria da drogas antimaláricas disponíveis, tornandose necessário pesquisar novos compostos com potencial atividade antimalárica. O objetivo desta tese foi inicialmente caracterizar a atividade do risedronato contra as formas intraeritrocitárias do parasita in vivo, além de identificar seu possível mecanismo de ação. A IC50 do risedronato foi de 20 mM em culturas de Plasmodium falciparum. Risedronato reduziu a biossíntese de FOH e GGOH e a isoprenilação de proteínas, inibindo a transferência do grupo FPP para as proteínas farnesiladas, entretanto, a transferência do GGPP para as proteínas geranilgeraniladas não foi inibida, isto também ocorreu quando proteínas ras e rab foram analisadas, sugerindo que a droga está inibindo a enzima FPPS. A enzima FPPS de P. falciparum foi expressa e obtivemos uma proteína recombinante fusionada a GST (rPfFPPS). Os substratos IPP, DMAPP, GPP e FPP foram utilizados para determinação da atividade catalítica da enzima, demonstrando FPP e GGPP como principais produtos. Os valores de Km para os diferentes substratos foi determinado. Demonstramos também que rPfFPPS é inibida por risedronato, podendo ser explorado como potencial alvo antimalárico.The increased resistance of the malaria parasite most of antimalarial drugs are available, making it necessary to search for new compounds with potential antimalarial activity. The aim of this thesis was initially characterize the activity of risedronate against intraerythrocytic forms of the parasite in vivo, and identify its possible mechanism of action. The IC50 of risedronate was 20 mM in cultures of Plasmodium falciparum. Risedronate reduced biosynthesis and FOH, GGOH and protein isoprenylation, inhibiting the transfer of FPP group for farnesylated proteins, however, the transfer of GGPP to geranygeranylated proteins was not inhibited, this also occurred when ras and rab proteins were analyzed, suggesting that the drug is inhibiting the enzyme FPPS. The FPPS enzyme from P. falciparum was expressed and obtained a recombinant protein fused to GST (rPfFPPS). The substrates IPP, DMAPP, GPP and FPP were used to determine the catalytic activity of the enzyme, demonstrating FPP and GGPP as main products. The Km values for the various substrates were determined. We also demonstrate that rPfFPPS is inhibited by risedronate, which can be exploited as potential antimalarial target.
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Gabriel, Heloisa Berti. "Caracterização da função biológica da vitamina K biossintetizada pelas formas intraeritrocitárias de Plasmodium falciparum." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-05082011-095206/.
Full textAbstract:
A falta de uma vacina eficaz e o problema da resistência aos fármacos têm dificultado o controle da malária. A busca de novos alvos biológicos para o desenvolvimento de antimaláricos eficazes tem se concentrado, em parte, na pesquisa e compreensão de vias metabólicas exclusivas do parasita. Nosso grupo vem investigando e caracterizando produtos da biossíntese de isoprenóides em P. falciparum. Resultados preliminares identificaram a biossíntese das duas formas da vitamina K: filoquinona (PhQ) e menaquinona (MQ), ambas provenientes das vias do chiquimato e da via 2-C-metil-D-eritritol-4-fosfato (MEP). Salienta-se, ainda, que as vias do chiquimato e MEP são exclusivas do parasita, portanto alvos interessantes para o estudo e desenvolvimento de drogas alternativas contra a malária. Ensaios enzimáticos demonstraram a participação da MQ-4 na cadeia respiratória como transportadora de elétrons. Resultados indicaram que o parasita controla a concentração de ubiquinona e menaquinona (UQ/MQ) de acordo com as condições de aeração a qual é submetido, assim como descrito em E. coli e Ascaris suum. A biossíntese de MQ em P. falciparum é bloqueada pelo composto Ro 48-8071, inibidor da enzima 1,4-dihidroxi-2-naftoato preniltransferase da via de biossíntese de MQ. Em relação a PhQ, dados preliminares mostram uma provável participação na proteção antioxidante no ciclo intraeritrocítico de P. falciparum. Finalmente, por meio de ensaios de Real Time-PCR, investigou-se o padrão de transcrição de prováveis genes que supostamente codificariam algumas enzimas da via de biossíntese de MQ, PhQ, e UQ (esse último previamente caracterizado). Os resultados demonstraram que não há alterações na transcrição desses genes prováveis nos parasitas mantidos em diferentes condições de pressão de O2.The lack of an effective vaccine and the problem of drug resistance haves hampered the control of malaria. The search for new biological targets for the development of effective antimalarials in part has focused on research and understanding of metabolic pathways unique to the parasite. Our group has investigated and characterized the products of the isoprenoids biosynthesis in P. falciparum. Preliminary results have identified the biosynthesis of two forms of vitamin K: phylloquinone (PhQ) and menaquinone (MQ), both derived from the Shikimate pathway and 2-C-methyl-D-erythritol-4-phosphate pathway (MEP). The shikimate and MEP pathways are unique to the parasite therefore are interesting targets for study and development of alternative drugs against the malaria. The enzimatic assay showed the participation of MQ-4 in the respiratory chain as electron carrier. Results indicated that the parasite controls the concentration of ubiquinone and menaquinone (UQ / MQ) according to the aeration conditions which is submitted, as described in E. coli and Ascaris suum. The MQ biosynthesis in P. falciparum is blocked by the compound Ro 48-8071, an inhibitor of the enzyme 1,4-dihydroxy-2-naftoato prenyltransferase. Also was described in the parasite, the biosynthesis of another form of vitamin K (PhQ) , and preliminary results showed probably participation of PhQ in the antioxidant protection in the cycle of P. falciparum. Finally, by the Real Time-PCR, we investigated the pattern of transcription of putative genes some enzymes of MQ, PhQ and UQ biosynthesis (the last was previously characterized). The results showed no changes in the transcription profile in the parasites kept in different conditions of O2 pressure.
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Häußler, Kristina Maria Elisabeth [Verfasser]. "Characterization and inhibition of NADPH-producing enzymes from the pentose phosphate pathway of Plasmodium parasites / Kristina Maria Elisabeth Häußler." Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1175873500/34.
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Häußler, Kristina [Verfasser]. "Characterization and inhibition of NADPH-producing enzymes from the pentose phosphate pathway of Plasmodium parasites / Kristina Maria Elisabeth Häußler." Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1175873500/34.
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Nascimento, Marília Nunes do. "Desenvolvimento de modelos de QSAR e planejamento de novos inibidores da enzima dUTPase de Plasmodium falciparum." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/5105.
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Malaria is a serious endemic disease caused by parasites of the genus Plasmodium, which affects much of the population, especially in tropical and subtropical areas. Currently, drug therapy makes use of artemisinin or its derivatives associated with a second anti-malarial drug. The shortage of new treatments as well as the spread of parasite resistance to drugs currently available, makes urgent the search and discovery of new targets and new antimalarial drugs. The enzyme deoxyuridine triphosphatase (dUTPase) of Plasmodium falciparum plays an important role in maintaining balance between 2'-deoxyuridine 5'-triphosphate (dUTP) and 2'- deoxitimina 5'-triphosphate (dTTP) in order to avoid the erroneous incorporation uracil on the DNA tape. Thus, the enzyme dUTPase is a potential target for the development of new drugs, and has been validated for the organisms Escherichia coli, Saccharomyces cerevisiae and Mycobacterium smegmatis. This study aimed to carry out quantitative studies of the relationship between structure and activity (QSAR) to a series of β-branched nucleoside inhibitors PfdUTPase, in order to generate robust and predictive models to predict compounds activity untested and that may help to elucidate the important structural requirements for the affinity of this class of compounds. For this, there was the hologram QSAR analysis (HQSAR), comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). For studies of CoMFA and CoMSIA were tested two methods of calculation of partial charges, the empirical method Gasteiger-Huckel and the semi-empirical method AM1-BCC. Were also tested three structural alignment strategies based on the binder: maximum common substructure, based on the overlap of molecular volumes, and on the basis of morphological similarities; and a strategy based on the 3D coordinates of the enzymeinhibitor complex (molecular docking). The QSAR models generated showed good robustness and external predictability, showing good power correlation and prediction of affinity. The HQSAR contribution maps and contour maps of the CoMFA and CoMSIA indicated the importance of certain groups for affinity, such as the importance of the presence of at least two of trityl rings that contribute both sterically as hydrophobically to interact with the hydrophobic site of the parasite enzyme, non-existent in the human enzyme. The drug design based on information obtained from 2D and 3D QSAR, generated 121 molecules grouped into 18 clusters. Two hits with approximate power to one of the most active compounds of the series stood out by presenting appropriate physicochemical properties.
A malária é uma doença endêmica grave, causada por parasitos do gênero Plasmodium, que afeta grande parte da população, em especial nas áreas tropicais e subtropicais. Atualmente, o tratamento farmacológico faz uso de artemisinina ou de seus derivados associado a um segundo fármaco antimalárico. A escassez de novos tratamentos assim como a disseminação da resistência do parasito aos fármacos atualmente disponíveis, torna urgente a busca e descoberta de novos alvos e novos fármacos antimaláricos. A enzima deoxiuridina trifosfatase (dUTPase) de Plasmodium falciparum desempenha um papel importante na manutenção do equilíbrio entre 2’-desoxiuridina 5’-trifosfato (dUTP) e 2’-deoxitimina 5’-trifosfato (dTTP), a fim de evitar a incorporação errônea de uracila na fita do DNA. Dessa forma, a enzima dUTPase é um alvo potencial para o desenvolvimento de novos fármacos, e já foi validada para os organismos Escherichia coli, Saccharomyces cerevisiae e Mycobacterium smegmatis. Este trabalho teve como objetivo a realização de estudos quantitativos de relação entre estrutura e atividade (QSAR) para uma série de nucleosídeos β-ramificados inibidores da PfdUTPase, com a finalidade de se gerar modelos robustos e preditivos para predizer a atividade de compostos não testados e que possam auxiliar na elucidação dos requisitos estruturais importantes para a afinidade desta classe de compostos. Para isso, realizou-se a análise de holograma QSAR (HQSAR), análise comparativa de campos moleculares (CoMFA) e a análise comparativa dos índices de similaridade molecular (CoMSIA). Para os estudos de CoMFA e CoMSIA, foram testados dois métodos de cálculo de cargas parciais, o método empírico Gasteiger-Huckel e o método semi-empírico AM1-BCC. Foram também testadas três estratégias de alinhamento estrutural baseadas no ligante: máxima subestrutura comum, baseada na sobreposição de volumes moleculares, e em função da similaridade morfológica; e uma estratégia baseada nas coordenadas 3D do complexo enzima-inibidor (docking molecular). Os modelos de QSAR gerados apresentaram boa robustez e preditividade externa, demostrando bom poder de correlação e predição da afinidade. Os mapas de contribuição de HQSAR e os mapas de contorno do CoMFA e CoMSIA indicaram a importância de determinados grupos para a afinidade, como por exemplo, a importância da presença de ao menos dois anéis tritila que contribuem tanto estericamente como hidrofobicamente para interação com o sítio hidrofóbico da enzima do parasito, inexistente na enzima de humanos. O planejamento de fármacos baseado nas informações obtidas do QSAR 2D e 3D, gerou 121 moléculas agrupadas em 18 clusters. Dois hits com potência aproximada a um dos compostos mais ativos da série se destacaram por apresentar propriedades físico-químicas apropriadas.
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Garcia, Mariana Lopes. "Estudos computacionais da enzima N-miristoiltransferase de Plasmodium falciparum e seus inibidores como candidatos a agentes antimaláricos." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-15092017-084415/.
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A malária é uma doença infecciosa causada pelos parasitas do gênero Plasmodium e transmitida pelo mosquito Anopheles spp. Devido ao surgimento de casos de resistência aos fármacos disponíveis novos alvos e candidatos a fármacos são necessários. Recentemente, a enzima N-miristoiltransferase (NMT) foi confirmada como essencial para o parasita e validada como alvo terapêutico para o desenvolvimento de candidatos a fármacos antimaláricos. O objetivo desse trabalho foi identificar os determinantes moleculares responsáveis pela atividade inibitória de uma série de derivados benzotiofênicos frente à NMT. Nesse sentido, estudos de relação quantitativa estrutura-atividade (QSAR) 2D e 3D foram desenvolvidos para dois conjuntos de dados de derivados benzotiofênicos como inibidores da enzima do parasita (PfNMT) e a homóloga humana (HsNMT). Além disso, estudos de modelagem por homologia da PfNMT foram conduzidos. Os estudos de QSAR 2D foram desenvolvidos pelo método de Holograma QSAR (HQSAR). O modelo estrutural de PfNMT foi aplicado na construção dos modelos QSAR 3D CoMFA (Comparative Molecular Field Analysis) e CoMSIA (Comparative Molecular Similarity Index Analysis). Os estudos de QSAR 3D foram conduzidos com diferentes métodos de cálculo de carga parcial atômica (Gasteiger-Hückel, MMFF94 e AM1-BCC, CHELPG e Mulliken) e de alinhamento molecular (Máxima Subestrutura Comum, alinhamento flexível e baseada no alvo molecular). Os melhores modelos construídos pelos métodos de QSAR 2D e 3D foram robustos, internamente consistentes e com elevada capacidade de predição da atividade de novos compostos contra a PfNMT. Os mapas de contribuição e de contorno geraram informações importantes sobre a relação estrutura-atividade dos compostos. Os resultados permitiram a identificação das bases moleculares responsáveis pela atividade dos inibidores benzotiofênicos e são úteis para o planejamento de novos inibidores mais potentes e seletivos para a enzima do parasita.Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium and transmitted by Anopheles spp. mosquitos. Due to the emerging resistance to current available drugs, great efforts for new molecular target and drugs are required. Recently, N-myristoyltransferase (NMT) was confirmed as an essential enzyme to malaria parasites and validated as a chemically tractable target for the development of new drug candidates against malaria. This work aimed to shed light on the molecular requirements underlying the inhibitory activity of benzothiophene derivatives against NMT. Therefore, 2D and 3D quantitative structure-activity relationship (QSAR) studies were developed for two datasets of benzothiophene derivatives as P. falciparum NMT (PfNMT) and the human homologue (HsNMT) inhibitors. Also, homology modeling studies for PfNMT were developed. The 2D QSAR studies were developed by the Hologram QSAR (HQSAR) method. The PfNMT structural model was applied in the construction of 3D QSAR models CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Index Analysis). Different molecular alignment (maximum common substructure, flexible alignment and structure based) and atomic partial charge calculation (Gasteiger-Hückel, MMFF94, AM1-BCC, CHELPG and Mulliken) methods were used to build the 3D QSAR models. The best models showed internal consistency and high predictive ability of biological activity against PfNMT. The contribution and contour maps gave important information about compounds structure-activity relationship. The results allowed the identification of the molecular requirements underlying the inhibitory activity and should be useful for the design of novel potent and selective PfNMT inhibitors as antimalarial drug candidates.
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Maluf, Fernando Vasconcelos. "Estudos estruturais e de química medicinal aplicados às enzimas da via glicolítica de protozoários: enolase de Plasmodium falciparum e gliceraldeído-3-fosfato desidrogenase de Trypanosoma cruzi." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-02102015-093453/.
Full textAbstract:
A melhor compreensão dos mecanismos fisiopatológicos e farmacológicos aliados a métodos modernos de investigação tornaram possível a descoberta e o desenvolvimento de fármacos para diversas doenças e disfunções orgânicas em humanos. Os fármacos desenvolvidos atualmente são resultados de intensos esforços em pesquisa por equipes multidisciplinares, impactando diretamente na qualidade de vida das diversas populações no mundo. Nesse cenário, os grupos de pesquisas estabelecidos em Universidades com foco no planejamento de fármacos para doenças tropicais têm crescido. A Malária e a Doença de Chagas figuram com especial importância, a primeira pela expressiva mortalidade mundial, enquanto a segunda pela morbidade e seus impactos na população brasileira. O tratamento de ambas possui limitações que se agravam, seja pelo baixo número de opções terapêuticas, ou pelo desenvolvimento de cepas resistentes. As enzimas investigadas nesse doutoramento, enolase (PfEnolase) de Plasmodium falciparum e gliceraldeído3fosfato desidrogenase de Trypanosoma cruzi (TcGAPDH), são componentes da via glicolítica destes parasitas e são considerados alvos moleculares atrativos para o desenvolvimento de inibidores enzimáticos, dada a importância destas enzimas no processo de obtenção de energia do parasita. Os estudos fundamentamse na busca por modulação seletiva da atividade biológica dos alvos selecionados através do desenvolvimento de novas moléculas bioativas. O estabelecimento de protocolo de expressão e purificação para enzima Pfenolase permitiu sua obtenção em quantidade e pureza suficiente para condução de estudos cinéticos e de triagem biológica, com a identificação de cinco novas classes químicas bastante promissoras; além de ensaios de cristalização, que culminaram na determinação da enzima em diversos complexos cristalográficos. Os dados estruturais produzidos foram fundamentais para condução da abordagem computacional de triagem virtual, que permitiu a identificação de 31 moléculas candidatas a inibidoras de Pfenolase. Avanços significativos foram obtidos também com a enzima TcGAPDH, destacando-se as adaptações nos processos de obtenção da proteína recombinante e ensaio cinético, condução de ensaio de bioprospecção orientada com a identificação e caracterização da molécula isolada (tilirosídeo). Novas condições de cristalização foram identificadas e poderão ser empregadas no processo de obtenção de complexos cristalográficos futuros. Adicionalmente, desenvolveu-se uma ferramenta computacional, Kinecteasy, para processamento automatizado dos dados produzidos das etapas de triagem biológica. Os trabalhos integrados de biologia estrutural e química medicinal desenvolvidos contribuem significativamente para o avanço no processo de planejamento de novos inibidores para as enzimas selecionadas.A better understanding of the pathophysiological and pharmacological mechanisms together with the modern research methods made possible the discovery and development of drugs for several humans´ diseases. The drugs currently developed are the result of intense efforts in research of multidisciplinary teams having as a direct consequence a remarkable impact on life quality of populations all over the world. In this scenario, research groups established at universities, with their focus on drug development for tropical diseases, are increasing. Malaria and Chagas disease deserve special attention, the former by the expressive world mortality, while the second by the morbidity and its impact on Brazilian population. Treatment for both has limitations, whether by the low number of therapeutic options, or by development of resistance. The target enzymes for this PhD project, enolase (PfEnolase) of Plasmodium falciparum and glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi (TcGAPDH), are essential components of glycolytic pathway and therefore related to the parasite energy production, thus, are considered attractive molecular targets for enzyme inhibitors development. Essentially, the proposed studies seek selective modulation of the target´s biological activity through the development of new bioactive molecules. The expression and purification protocols developed for Pfenolase have allowed us to obtain recombinant protein at suitable yield and purity for conducting screening assays, which has revealed five new chemical classes as Pfenolase inhibitors. Crystallization experiments were successfully conducted and 3D structure were determined for different complexes. Structural data was essential for performing the computational approach of virtual screening, which has allowed us to identify 31 inhibitor candidates for Pfenolase. Significant advances were obtained with TcGAPDH, highlighting the adaptations on recombinant protein protocol and kinetic assay. Assay-guided bioprospecting experiments were successfully performed with identification and characterization of isolated inhibitor (tiliroside). New crystallization conditions were identified and will be employed in future co-crystallization and soaking studies. Additionally, Kinecteasy, a computational tool, were developed for automated data processing of biological screening assays. The structure and medicinal chemistry studies presented here contribute significantly in the process of drug development for the selected enzymes.
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Macedo, Karlla Gonçalves de. "Estudos de SAR e QSAR para um conjunto de triazolopirimidinas inibidores da enzima diidroorotato desidrogenase de Plasmodium falciparum." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/4755.
Full textAbstract:
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Drug discovery and development process requires high investments of both time and money. Strategies for drug design aided by computers, CADD (Computer-Aided Drug Design) have gained prominence over the last decades, in order to minimize the impact of those costs. CADD techniques also allow the exploration of a greater number of biological targets and promising molecules. Malaria is an endemic disease in Africa and in South American caused by the protozoa of the genus Plasmodium. In 2012, 207 million cases and 627,000 deaths were estimated, according to the World Health Organization. The enzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step of the pyrimidine biosynthesis, and consists in a validated target for the design of new antimalarial agents. The aim of this study was to develop structure-activity relationships (SAR) rules and to generate quantitative structure-activity relationships (QSAR) models using a set of triazolopyrimidines described in the literature as inhibitors of DHODH from P. falciparum (PfDHODH). SAR rules were established using methods of clustering, activity cliffs and activity landscapes. In addition, several models of 2D-QSAR and hologram QSAR (HQSAR) were developed and validated. The SAR analyses allowed the understanding of the basic structural requirements for the antimalarial activity of triazolopyrimidines, like alkyl halides substituents on the triazolopimidinic ring, hydrophobic substituents in the para position on the benzene ring, all in agreement with the chemical space inside the active site of the PfDHODH. The HQSAR and 2D-QSAR models showed good statistical parameters and good predictive ability. The HQSAR contour maps were also consistent with the chemical space of the active site of the enzyme. The results of this study could serve as guide for the design of new antimalarials with higher potency.
O processo de planejamento e desenvolvimento de novos fármacos é um trabalho complexo, que demanda elevados investimentos de tempo e dinheiro. Estratégias de planejamento de fármacos auxiliadas por computador, CADD (Computer-Aided Drug Design) vêm se destacando, pois minimizam gastos e tempo, além de poder explorar um número maior de alvos biológicos e moléculas promissoras. A malária é uma doença endêmica grave na África e América do Sul, causada por protozoários do gênero Plasmodium. Em 2012 foram estimados 207 milhões de casos e 627.000 mortes, de acordo com a Organização Mundial da Saúde. A enzima diidroorotato desidrogenase (DHODH) atua na quarta etapa da biossíntese de pirimidinas, é um alvo validado para o planejamento de novos agentes antimaláricos. O objetivo geral deste trabalho foi desenvolver regras de relação entre estrutura e atividade (SAR) e modelos robustos e preditivos de relações quantitativas entre estrutura e atividade bidimensionais (QSAR-2D), utilizando um conjunto de triazolopirimidinas descritas na literatura como inibidores da DHODH de P. falciparum (PfDHODH). Foram desenvolvidas regras de SAR utilizando os métodos de análise de agrupamentos, cliffs de atividade e landscapes de atividade. Além disso, desenvolveu-se e validou-se vários modelos de QSAR–2D e de holograma QSAR (HQSAR). As análises de SAR, permitiram estabelecer requisitos estruturais essenciais para a atividade antimalárica das triazolopirimidinas, como substituintes haletos de alquila no anel triazolopimidínico, substituintes hidrofóbicos na posição para no anel benzênico, todos de acordo com o espaço químico da cavidade de interação da PfDHODH. Os modelos de HQSAR e QSAR-2D apresentaram bons parâmetros estatísticos e boa capacidade preditiva. Os mapas de contribuição de HQSAR também estão de acordo com o espaço químico da cavidade de interação da PfDHODH. Os dados obtidos servem como guia para o planejamento de novos antimaláricos com maior potência.
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Moyo, Sipho Dugunye. "Comparative study of clan CA cysteine proteases: an insight into the protozoan parasites." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1020309.
Full textAbstract:
Protozoan infections such as Malaria, Leishmaniasis, Toxoplasmosis, Chaga’s disease and African trypanosomiasis caused by the Plasmodium, Leishmania, Toxoplasma and Trypanosoma genuses respectively; inflict a huge economic, health and social impact in endemic regions particularly tropical and sub-tropical regions. The combined infections are estimated at over a billion annually and approximately 1.1 million deaths annually. The global burden of the protozoan infections is worsened by the increased drug resistance, toxicity and the relatively high cost of treatment and prophylaxis. Therefore there has been a high demand for new drugs and drug targets that play a role in parasite virulence. Cysteine proteases have been validated as viable drug targets due to their role in the infectivity stage of the parasites within the human host. There is a variety of cysteine proteases hence they are subdivided into families and in this study we focus on the clan CA, papain family C1 proteases. The current inhibitors for the protozoan cysteine proteases lack selectivity and specificity which contributes to drug toxicity. Therefore there is a need to identify the differences and similarities between the host, vector and protozoan proteases. This study uses a variety of bioinformatics tools to assess these differences and similarities. The Plasmodium cysteine protease FP-2 is the most characterized protease hence it was used as a reference to all the other proteases and its homologs were retrieved, aligned and the evolutionary relationships established. The homologs were also analysed for common motifs and the physicochemical properties determined which were validated using the Kruskal-Wallis test. These analyses revealed that the host and vector cathepsins share similar properties while the parasite cathepsins differ. At sub-site level sub-site 2 showed greater variations suggesting diverse ligand specificity within the proteases, a revelation that is vital in the design of antiprotozoan inhibitors.
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Leoratti, Fabiana Maria de Souza. ""Resposta imune humoral na malária humana: quantidade e qualidade de anticorpos anti-Plasmodium falciparum"." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-04102005-144150/.
Full textAbstract:
Neste estudo avaliamos a resposta imune humoral de indivíduos naturalmente expostos à malária em áreas endêmicas no Brasil. Os anticorpos IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE e IgA anti-formas eritrocitárias de Plasmodium falciparum foram determinadas por ELISA. Anticorpos IgG, IgG1, IgG2 de alta avidez e IgG3 de baixa avidez predominaram nos indivíduos sem complicações de malária ou assintomáticos, enquanto anticorpos IgG4, IgE e IgM predominaram nos indivíduos com complicações clínicas por malária. Os resultados mostram que mesmo em regiões com transmissão instável de malária pode ser observado o desenvolvimento de imunidade protetora quando anticorpos apropriados são produzidosIn this study, we have evaluated the humoral immune response of individuals naturally exposed to malaria living in endemic areas of Brazil. We determined IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA antibodies against Plasmodium falciparum blood stages by ELISA. We observed that the level of high avidity IgG, IgG1 and IgG2 and low avidity IgG3 antibodies were higher in asymptomatic individuals or with uncomplicated malaria, while IgG4, IgE and IgM antibodies were higher in individuals with complicated malaria. Taken together the results showed that even in unstable malaria regions it can be observed the development of protective immunity against malaria when appropriate antibodies are produced
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Grava, Andréa Fagundes. "Utilização da enzima β-cetoacil ACP redutase (OAR) da via FAS II de Plasmodium falciparum como um alvo para busca de novos compostos antimaláricos." Universidade Federal do Amazonas, 2014. http://tede.ufam.edu.br/handle/tede/5657.
Full textAbstract:
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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Malaria is a parasitic disease with the highest number of hospitalizations and deaths each year worldwide, one of the major public health problems in Africa, South America and East Asia. According to estimates by the World Health Organization, 198 million people became ill in 2014. This evidence led to the development of new strategies for the treatment of malaria in order to minimize the growing problem of parasite resistance to commonly used medicinal products. With the elucidation of metabolic pathways essential for some of the malaria parasite development, new molecular targets have been proposed for the development of new drugs, among which can be cited Hypoxanthine Guanine Phosphoribosyltransferase (HGPRT) Chorismate synthase, enoyl-ACP reductase and purine nucleoside phosphorylase (PNP). Objective: The objective of this study was the identification of compounds as potential inhibitors of beta-ketoacyl-ACP reductase enzyme - OAR Plasmodium falciparum. Results: The 28kDa enzyme was expressed in Escherichia coli and purified by affinity chromatography. Searches of new molecules through Sceenning by Virtual Docking were able to find 30 molecules of which was selected Skyrin molecule due to its higher affinity for the enzyme OAR. Analysis of enzyme activity by spectrophotometry using NADPH and acetoacetyl-CoA substrates the enzyme showed activity OAR reducing NADPH in the various substrates concentrations, and was shown to be inhibited by Skyrin. It was also possible to show by surface plasmon resonance (SPR) Skyrin that the molecule binds to the enzyme OAR but dissociates easily.In tests using cell cultures infected with P. falciparum this molecule showed antimalarial activity with IC50 8,88μg / ml, and is not toxic to HepG2 cell line. Conclusion:This new approach has significant advantages compared to traditional methods, since it establishes in advance the specific bioactive and its mechanism of action. Ideal targets for the development of antimalarial drugs against infectious agents must be essential to the survival of the pathogen and the host is absent.
A Malária é a doença parasitária com maior número de internações e mortes por ano em todo o mundo, sendo um dos maiores problemas de saúde pública na África, América do Sul e Ásia Oriental. Segundo estimativas da Organização Mundial de Saúde, 198 milhões de pessoas ficaram doentes em 2014. Essas evidências levaram ao desenvolvimento de novas estratégias para o tratamento da malária a fim de minimizar o crescente problema da resistência do parasita aos medicamentos de uso corrente. Com a elucidação de algumas Vias Metabólicas essenciais para do desenvolvimento do parasita da malária, novos alvos moleculares foram propostos para o desenvolvimento de novas drogas, entre eles pode-se citar a Hipoxantina Guanina Fosforribosiltransferase (HGPRT), Corismato sintase, Enoil –ACP redutase e a Fosforilase de nucleosídeos purínicos (PNP). Objetivo: O objetivo desse estudo foi a identificação de compostos como possíveis inibidores contra a enzima Beta-cetoacil- ACP- redutase - OAR de Plasmodium falciparum. Resultados: A enzima de 28kDa, foi expressa em Escherichia coli e purificada através de cromatografia de afinidade. Buscas de novas moléculas através de Sceenning Virtual por Docking foram capazes de encontrar 30 moléculas das quais foi selecionada a molécula Skyrin por apresentar maior afinidade pela enzima OAR. Análise da atividade enzimática por espectrofotometria utilizando os substratos NADPH e Acetoacetil-COA a enzima OAR apresentou atividade reduzindo NADPH, em concentrações dos substratos variadas, e mostrou ser inibida por Skyrin. Foi possível mostrar também por Ressonância Plasmônica de Superfície (SPR) que a Skyrin liga-se à enzima OAR porém dissocia-se facilmente. Em testes utilizando-se culturas celulares infectadas com P. falciparum essa molécula apresentou atividade antimalárica com IC50 de 8,88μg/mL, não sendo tóxica para a linhagem celular HepG2. Conclusão: Esta nova abordagem apresenta vantagens significativas quando comparada aos métodos tradicionais, uma vez que se estabelece antecipadamente a especificidade do bioativo e respectivo mecanismo de ação. Alvos ideais para o desenvolvimento de fármacos contra agentes infecciosos antimaláricos devem ser essencial para a sobrevivência do patógeno e estar ausente no hospedeiro.
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VALLONE, ALESSANDRA. "INVESTIGATION OF NOVEL THERAPEUTIC TOOLS AGAINST INFECTIOUS DISEASES Part 1. Medicinal Chemistry Investigation of MMV019918 Derivatives as Dual Schizonticide And Gametocytocidal Agents Against Plasmodium falciparum Part 2. Investigation of 5-Aryl-Heterocycles As Potential Inhibitors of Metallo beta-Lactamase Enzymes." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1004943.
Full textAbstract:
Among infectious diseases, two large groups have great clinical relevance: parasitic and bacterial
infections. Belonging to the first category is malaria, caused by the parasite Plasmodium
falciparum. Transmission of Plasmodium parasites between humans and Anopheles mosquitoes is
one of the most important contributors to the global impact of malaria and to the difficulties
encountered in eliminating this parasite1
. Gametocytogenesis, the process by which merozoites
switch from asexual replication to produce male and female gametocytes, represents a critical step
in malaria transmission and Plasmodium genetic diversity. Still too little is known about the
biochemical events that regulate gametocytogenesis and there are few existing drugs able of
inhibiting the gametocytes development and block malaria transmission2
. To encourage drug
discovery and research, the non-profit foundation Medicines for Malaria Venture (MMV) has
provided a library of 400 compounds that present antimalarial activity in the micromolar range, but
their molecular targets and mode of action are not necessarily known3
. Here we describe the
medchem investigation of one of the most promising hit compound included in this library,
MMV019918. MMV019918 has been highlighted in several in vitro studies for its promising antigametocyte
activity coupled to activity against schizonts. On the basis of its structure, the synthesis
of a new series of compounds with transmission-blocking activity has been designed. It has been
found very interesting the activity of one derivative NF2350 which resulted active also in the
standard membrane feeding assay (SMFA), to measure subsequent mosquito infection, and which
has an improved toxicological profile compared to MMV019918. A further investigation of NF2350
will allow us to optimize the transmission-blocking activity and to indentify its putative target.
Regarding bacterial infections, a critical issue to be addressed is bacterial resistance to antibiotics
and in particular to β-lactams. Metallo-β-lactamases (MBL) are a family of enzymes involved in the
widespread mechanisms of resistance to beta-lactam antibiotics, The diffusion of MBL-producing
isolates of Pseudomonas aeruginosa, a bacterial pathogen of primary relevance for both
nosocomial and chronic infections of the respiratory tracts in cystic fibrosis patients, is notably
increasing in some specific settings. No clinically useful MBLs inhibitors are currently available in
therapy3
Here we will present the design, synthesis, and biological investigation of a series of
functionalized 2-arylfuran compounds with sub-micromolar antiplasmodial activity, against both
asexual and sexual stages. Furthermore, appropriate decoration of this molecular scaffold allowed
to obtain activity against some isoforms of MBL (including IMP-1and NDM-1).
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Tello, Sánchez Maribel Liliana. "Eficacia del ensayo inmuno-enzimático de detección de la enzima lactato deshidrogenasa (Deli) y ensayo de fluorescencia para malaria basado en el reactivo SYBR green-I (MSF) para calcular la IC50 de drogas anti-Plasmodium falciparum. Iquitos 2015." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2019. https://hdl.handle.net/20.500.12672/10853.
Full textAbstract:
Determina la eficacia de las pruebas DELI y MSF para calcular la IC50 de drogas antimaláricas mefloquina, quinina y cloroquina obtenidas de aislamientos de P. falciparum provenientes de pobladores de la comunidad de Padre Cocha en Iquitos-Perú. Se realizó un estudio cuantitativo descriptivo, prospectivo de corte transversal. La muestra fueron 16 muestras de sangre con diagnóstico de malaria confirmado por gota gruesa. Se realizaron los dos ensayos de sensibilidad in vitro (DELI y MSF) a cada muestra. Se determinaron tres factores de eficacia para el presente estudio; porcentaje de éxito, coeficiente de determinación de curva (R2) y coeficiente de variación (CV). Se hizo un análisis descriptivo y estadístico de los factores de eficacia mediante las pruebas de Wilcoxon y McNemar- Bowker para muestras pareadas con p < 0.05. Las medias aritméticas de los valores de IC50 con el ensayo DELI fueron para cloroquina 231.26 nM, quinina 101.17 nM y mefloquina 16.03 nM. Las medias de los valores de IC50 con el ensayo MSF fueron para cloroquina 227.52 nM, quinina 142.46 nM y mefloquina 35.07 nM. El porcentaje de éxito del cálculo de la IC50 para las tres drogas fueron el 50% (8/16) y 87.5% (14/16) en los ensayos MSF y DELI respectivamente, estas diferencias fueron estadísticamente significativas (p < 0.05). Sin embargo, en el análisis entre los porcentajes de éxito entre drogas, no presentaron diferencias para CQ y QN y si presentaron diferencias para MQ (p < 0.05). No hay diferencias significativas entre los valores de R2 entre las pruebas MSF y DELI. El porcentaje de éxito de CV positivos aumentó de 37.5% con el ensayo MSF a 81.25% con el ensayo DELI, estas diferencias fueron significativas (p < 0.05). Se concluye que el ensayo DELI es más eficaz que el ensayo MSF para calcular la IC50 de las drogas CQ, QN Y MQ.Tesis
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Banerjee, Mousumi. "Structure-Function Studies On Triosephoshate Isomerase From Plasmodium falciparum And Methanocaldococcus jannaschii." Thesis, 2008. http://hdl.handle.net/2005/824.
Full textAbstract:
This thesis describes studies directed towards understanding structure-function relationships of triosephosphate isomerase (TIM), from a protozoan parasite Plasmodium falciparum and a thermophilic archaea Methanocaldococcus jannaschii. Triosephosphate isomerase, a ubiquitous glycolytic enzyme, has been the subject of biochemical, enzymatic and structural studies for the last five decades. Studies on TIM have been central to the development of mechanistic enzymology. The present study investigates the role of specific residues in the structure and function of Plasmodium falciparum triosephosphate isomerase (PfTIM). The structure and stability of a tetrameric triosephosphate isomerase from Methanocaldococcus jannaschii (MjTIM) is also presented.
Chapter 1 provides a general introduction to the glycolytic enzyme triosephosphate isomerase, conservation of TIM sequences, its fold and three dimensional organization. The isomerisation reaction interconverting dihydroxyacetone phosphate and glyceraldehyde 3phosphate catalyzed by triosephosphate isomerase is an example of a highly stereospecific proton transfer process (Hall & Knowles, 1975; Rieder & Rose, 1959). This chapter briefly reviews mechanistic features and discusses the role of active site residues and the functional flexible loop 6. Triosephosphate isomerase adopts the widely occurring ( β/ α)8 barrel fold and mostly occurs as a dimer (Banner et al., 1975). Protein engineering studies, related to folding, stability and design of monomeric TIM are also addressed. A brief introduction to thermophilic TIMs and higher oligomeric TIMs is given. The role of this enzyme in disease states like hemolytic anemia and neuromuscular dysfunction is surveyed. The production of methylglyoxal, a toxic metabolite, as a byproduct of the TIM reaction is also considered.
Many proteins utilize segmental motions to catalyze a specific reaction. The omega loop (loop 6) of triosephosphate isomerase is important for preventing the ene-diol intermediate from forming the cytotoxic byproduct, methylglyoxal. The active site loop-6 of triosephosphate isomerase moves about 7Ǻ on ligand binding. It exhibits a hinged lid motion alternating between two well defined, “open” and “closed”, conformations (Joseph et al., 1990). Though the movement of loop 6 is not ligand gated, in crystals the ligand bound forms invariably reveal a closed loop conformation. Plasmodium falciparum TIM is an exception which predominantly exhibits “open” loop conformations, even in the ligand bound state (Parthasarathy et al., 2002). Phe 96 is a key residue that is involved in contacts between the flexible loop-6 and the protein body in PfTIM. Notably, in all TIM sequences determined thus far, with the exception of plasmodial sequences, this residue is Ser 96. In Chapter 2 the mutants F96S, F96H and F96W are reported. The crystal structures of the mutant enzymes with or without bound ligand are described. In all the ligand free cases, loop-6 adopts an “open” conformation. Kinetic parameters for all the mutants establish that residue 96 does not play an essential role in modulating the loop conformation but may be important for ligand binding. Structural analysis of the mutants along with WT enzyme reveals the presence of a water network which can modulate ligand binding.
Subunit interfaces of oligomeric proteins provide an opportunity to understand protein- protein interactions. Chapter 3 describes biochemical and biophysical studies on two separate dimer-interface destabilizing mutants C13E and W11F/W168F/Y74W of PfTIM. The intention was to generate a stable monomer by disrupting the interaction hubs. C13 is a part of a large hydrophobic patch (Maithal et al., 2002a) at the dimer interface. Introduction of a negative charge at position 13 destabilizes the interface and reduces activity. Y74 is a part of an aromatic cluster of the interface (Maithal et al., 2002b). The Y74W triple mutant was designed to disrupt the aromatic cluster by introducing additional atoms. Tryptophan is also a fluorophore, allowing studies of the dimer disruption by fluorescence, after mutating the two inherent tryptophan residues, W11 and W168 to phenylalanine. The mutants showed reduced activity and were more sensitive than the wild type enzyme to chemical denaturants as well as thermal denaturation. Evidenced for monomer formation is presented. These studies together with previous work reveal that the interface is important for both activity and stability.
In order to develop a model for understanding the relationship between protein stabilization and oligomeric status, characterization of the TIM from Methanocaldococcus jannaschii (MjTIM) has been undertaken. Chapter 4 describes the purification and characterization of MjTIM. The MjTIM gene was cloned and expressed in pTrc99A and protein was isolated from AA200 E. coli cells. Hyperexpressed protein was purified to homogeneity and relevant kinetic parameters have been determined. The tetrameric nature of MjTIM is established by gel filtration studies. Circular dichroism (CD) studies establish the stability of the overall fold, even at temperatures as high as 95ºC. A surprising loss of enzyme activity upon prolonged incubation at high temperature was observed. ESI-MS studies establish that oxidation of thiol groups of the protein may be responsible for the thermal inactivation.
Chapter 5 describes the molecular structure of MjTIM, determined in collaboration with Prof. MRN Murthy’s group at the Indian Institute of Science (Gayathri et al., 2007). The crystal structure of the recombinant triosephosphate isomerase (TIM) from the archaeabacteria Methanocaldococcus jannaschii has been determined at a resolution of 2.3 Å. MjTIM is tetrameric, as suggested by solution studies and from the crystal structure, as in the case of two other structurally characterised archaeal TIMs. The archaeabacterial TIMs are shorter compared to the dimeric TIMs, with the insertions in the dimeric TIMs occurring in the vicinity of the putative tetramer interface, resulting in a hindrance to tetramerization in the dimeric TIMs. The charge distribution on the surface of archaeal TIMs also facilitates tetramerization. Analysis of the barrel interactions in TIMs suggests that these interactions are unlikely to account for the thermal stability of archaeal TIMs. A feature of the unliganded structure of MjTIM is the complete absence of electron density for the loop 6 residues. The disorder of the loop may be ascribed to a missing salt bridge between residues at the N- and C- terminal ends of the loop in MjTIM.
Chapter 6 is a follow up of an interesting observation made by Vogel and Chmielewski (1994), who noticed that subtilisin cleaved rabbit muscle triosephosphate isomerase religated spontaneously upon addition of organic solvents. Further extension of this nicking and religation process with PfTIM emphasizes the importance of tertiary interactions in contributing to the stability of the (β/α)8 barrel folds (Ray et al., 1999). This chapter establishes that subtilisin nicking and religation is also facile in thermophilic MjTIM. Fragments generated by subtilisin nicking were identified using MALDI mass spectrometry at early and late stages of the cleavage for both the dimeric PfTIM and tetrameric MjTIM. This chapter also describes the comparative thermal and denaturant stability of both the enzymes. The accessibility of the Cys residues of MjTIM has been probed by examining the rates of labeling of thiol groups by iodoacetamide. The differential labeling of Cys residues has been demonstrated by mass spectrometry.
Chapter 7 summarizes the main results and conclusions of the studies described in this thesis.
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Marreiros, Maria Inês Moreira Oliveira Leite. "Characterization of Plasmodium methionine metabolism key enzyme." Master's thesis, 2016. http://hdl.handle.net/10451/25946.
Full textAbstract:
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016Malaria is a disease caused by protozoan parasites of the genus Plasmodium that are transmitted to humans by infected female Anopheles mosquitoes. Despite countless efforts toward eradication malaria still remains one of the most prevalent infectious diseases, constituting a major public health concern. The available antimalarial drugs are insufficient to control and eradicate malaria, mostly due to the emergence of drug-resistant parasites. Thus, the development of novel intervention strategies is critical to achieve eradication. As an obligatory intracellular pathogen, Plasmodium establishes close interactions with its host that are crucial to ensure parasite development and survival, one of such is the methionine metabolism. Methionine is an essential amino acid and, as for most living organisms, Plasmodium lacks the ability to synthesize methionine de novo. During the blood-stage of infection Plasmodium obtains methionine mainly through haemoglobin digestion. However, how Plasmodium obtains methionine during the liver-stage and how the parasite modulates the host cells in order to scavenge this essential amino acid is still unknown. The first step of methionine cycle is the synthesis of S-adenosylmethionine (SAMe) through a reaction catalyzed by the enzyme SAMe synthetase (SAMS). SAMe is a key metabolite in the methionine metabolism being the main biological donor of methyl groups for transmethylation reactions. SAMe is also a key intermediate in the transsulfuration pathway generating homocysteine (Hcy) which is metabolized into glutathione (GSH), being the last step of this pathway catalysed by glutathione synthetase (GS). GSH is a powerful antioxidant that in Plasmodium acts as one of the primary lines of the defense against the damage caused by reactive oxygen species (ROS), ensuring parasite survival. In this work we have explored the role of Plasmodium enzymes responsible for SAMe and GSH synthesis throughout its life cycle and in particular during the liver-stage of infection. The liver is a particular organ in the metabolism of methionine, namely in SAMe-dependent transmethylation reactions and in glutathione synthesis and storage. Thus, we hypothesized that while replicating inside hepatocytes, Plasmodium relies on its host to ensure a sufficient supply of these crucial metabolites. The data obtained in this study suggest that: 1) Plasmodium does not rely on its own SAMS enzyme while developing inside hepatocytes; 2) that the inhibition of SAMS activity during the blood-stage of infection leads to a low parasitemia, preventing the onset of cerebral malaria and 3) the deletion of GS-encoding gene results in the arrest at the oocyst stage, preventing transmission between the mosquito vector and the mammalian host. A detailed knowledge of Plasmodium methionine pathway provides promising tools for the design and development of novel antimalarial drugs.
A malária é uma doença causada por parasitas protozoários pertencentes ao género Plasmodium que são transmitidos aos humanos por mosquitos fêmea do género Anopheles. Apesar dos inúmeros esforços realizados na tentativa de erradicar a malária esta permanece ainda uma das doenças parasíticas mais prevalentes, constituindo um problema de saúde público. Os anti-maláricos disponíveis são insuficientes no controlo e erradicação da malária, devido sobretudo ao aparecimento de parasitas resistentes. Além disso, o escasso conhecimento acerca da biologia do parasita bem como das interações que este estabelece com o hospedeiro constituem uma barreira na luta contra a malária. Assim, o desenvolvimento de novas estratégias de intervenção torna-se crucial para conseguir a erradicação. Plasmodium é um patogénio intracelular obrigatório e, como tal, as interações que estabelece com o seu hospedeiro são essenciais para garantir o seu desenvolvimento e sobrevivência, nomeadamente as que estabelece ao nível do metabolismo da metionina. A metionina é um aminoácido essencial pelo que, tal como na maioria dos organismos, Plasmodium não tem capacidade para a sintetizar de novo. Durante a fase sanguínea Plasmodium obtém metionina maioritariamente através da degradação de hemoglobina. Contudo, os mecanismos que Plasmodium utiliza para obter metionina durante a fase hepática, bem como para modular a célula hospedeira de modo a garantir um fornecimento suficiente deste aminoácido são ainda desconhecidos. O primeiro passo do ciclo da metionina consiste na síntese de S-adenosilmetionina (SAMe) numa reação catalisada pela enzima SAMe sintetase (SAMS). A SAMe é um metabolito essencial na via metabólica da metionina sendo o maior dador biológico de grupos metilo. A SAMe é ainda um importante intermediário na via da transsulfuração sendo convertida em homocisteína e subsequentemente metabolizada em glutationo, sendo o último passo desta via catalisado pela glutationo sintetase (GS). O glutationo é um antioxidante que em Plasmodium atua como uma das primeiras linhas de defesa contra espécies oxidativas. Neste trabalho explorámos o papel das enzimas de Plasmodium responsáveis pela síntese de SAMe e glutationo ao longo do seu ciclo de vida, com particular ênfase na fase hepática da infeção. O fígado tem um papel preponderante no metabolismo da metionina, nomeadamente nas reações de transmetilação dependentes de SAMe bem como na regulação da síntese e armazenamento do glutationo. Assim, a hipótese que propusemos testar é que enquanto replica no interior do hepatócito Plasmodium depende do hospedeiro para garantir a obtenção destes metabolitos essenciais. Os resultados obtidos neste estudo demonstram que: 1) durante o seu desenvolvimento no fígado Plasmodium não depende da atividade da sua enzima SAMS; 2) a inibição da atividade da enzima SAMS durante a fase sanguínea da infeção resulta numa redução da parasitémia, prevenindo o aparecimento de malária cerebral e ainda que; 3) a deleção do gene que codifica para a enzima GS inibe o desenvolvimento dos esporozoítos, bloqueando assim a transmissão entre o vetor e o hospedeiro mamífero. Assim, um conhecimento detalhado do metabolismo da metionina em Plasmodium fornece ferramentas promissoras para o desenvolvimento de novos anti-maláricos.
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Maity, Koustav. "Structure Analysis Of FabI And FabZ Enzymes Of The Fatty Acid Biosynthesis Pathway Of Plasmodium Falciparum." Thesis, 2010. http://hdl.handle.net/2005/2221.
Full textAbstract:
The emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease.
The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study.
Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors.
Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.
The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms.
The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.
Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies.
Part of the work presented in the thesis has been reported in the following publications.
Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9.
Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37.
Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91.
Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76.
Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated)
Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated)
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Pidugu, Lakshmi Swarna Mukhi. "Structural Studies On The Enzymes FabI And FabZ Of Plasmodium Falciparum." Thesis, 2006. http://hdl.handle.net/2005/381.
Full textAbstract:
The thesis deals with X-ray crystallographic analysis of two enzymes involved in the fatty
acid biosynthesis pathway, known as Fatty Acid Synthase or FAS, of the malarial parasite, Plasmodium falciparum, in order to understand their functions at the atomic level and to provide structural basis for the rational design of antimalarial compounds. Targeting highly specific and well-characterized biochemical pathways to develop effective therapeutic agents has the advantage of designing new drugs or modifying the existing ones based on the details of the known features of the processes. Knowledge of the three-dimensional structures of the molecules involved in the reactions will enhance the capabilities of this procedure.
The recently identified fatty acid biosynthesis pathway in Plasmodium falciparum is undoubtedly an attractive target for drug development as it differs from that in humans. In the malarial parasite, each reaction of the pathway is catalyzed by a specific enzyme whereas in humans, the synthesis is carried out by a single multidomain enzyme. Essentially each step in the FAS of P. falciparum can be a potential target to prevent the growth of the parasite as the fatty acids are essential for the formation of the cell membrane which is vital for its survival. All
the reactions of this pathway have been well-characterized. Nevertheless, there is a dearth of structural information of the proteins involved in performing various functions in this pathway. When this work was initiated, crystal structures of none of these proteins were reported. The current work on the plasmodial FAS proteins has been undertaken with a view to obtain precise
structural details of their reaction and inhibition mechanisms.
The introductory chapter of the thesis includes a discussion on malaria, the fatty acid biosynthesis in various organisms and an overview of the structural features of the enzymes involved in the pathway that have been characterized from other organisms.The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the other computational and biophysical methods used to further characterize the enzymes under study.
FabI, the enoyl acyl carrier protein reductase, that regulates the third step in FAS has been crystallized as a binary complex with its cofactor NADH and as a ternary complex with NAD+and triclosan. The crystal structures of the binary and the ternary complexes have been determined at 2.5 and 2.2 ˚A, respectively. The mode of binding of the cofactor and the inhibitor triclosan to the enzyme with details of the interactions between them could be clearly
obtained from these structures. Each subunit of the tetrameric FabI has the classical Rossmann fold. We carried out a thorough analysis of this structure and compared it with the FabI structures from various sources, four microbial (Escherichia coli, Mycobacterium tuberculosis and Helicobacter pylori) and one plant (Brassica napus), and arrived at a number of significant conclusions: Though the tertiary and the quaternary structures of the enzymes from different sources are similar, the substrate binding loop shows significant changes. The position and nature of the loop are strongly correlated to the affinity of triclosan to the enzyme. Small to major changes in the structure of the enzyme occur to enhance ligand binding. Water molecules play an important role in enzyme-ligand interactions. The crystal structure has also confirmed our previous prediction based on modeling studies of the enzyme that the introduction of bulkier groups at carbon 4’ of triclosan is likely to improve its efficacy as an inhibitor of FabI of P.
falciparum. It has also provided the structural basis for its preference to bind to the coenzyme NADH but not to NADPH which was also predicted by our modeling studies. Chapters 3 and 4 discuss the structure solution and a comparative analysis of the crystal structures of FabIs from various sources.
The crystal structure of FabZ, the β-hydroxyacyl acyl carrier protein dehydratase of P. falciparum, has been determined at a resolution of 2.4 ˚A. Each subunit of FabZ has a hotdog fold with one long central α-helix surrounded by a six-stranded antiparallel β-sheet. FabZ has been found to exist as a homodimer in the crystals of the present study in contrast to the hexameric form which is a trimer of dimers crystallized in a different condition, reported while we completed the structure of the dimeric form. In the dimeric form, the architecture of the catalytic site has been drastically altered with two catalytic histidine residues moving away from the catalytic site caused by two cis to trans peptide flips compared to the hexameric form. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate-binding. The dimeric state and an altered catalytic site architecture make the dimeric FabZ presented in the thesis distinctly
different from the FabZ structures described so far. This is the first observation and report of the existence of an inactive form of the enzyme and its unique structural features. Further analysis using dynamic light scattering and size exclusion chromatographic studies have shown that a
pH-related conformational switching occurs between the inactive dimers and active hexamers of FabZ in P. falciparum. These findings open alternate and entirely new strategies to design inhibitors to make FabZ inactive. FabZ crystals show polymorphism with varying length of its longest cell axis. We could collect X-ray diffraction data for three of these forms. An analysis
of these forms revealed that three flexible loops of the structure including those containing the peptide flips compete for the space between two symmetry-related molecules. In the form with the longest cell axis, the loops have the highest stability resulting in a better diffraction from
the crystal. We also performed docking studies with two previously characterized inhibitors of FabZ. The docking showed that the inhibitors bind strongly at the active site each one making a number of different interactions with the catalytic residues. Observations from our docking studies are in excellent agreement with and strongly supported by chemical modification and
fluorimetric analysis of the wild type enzyme and its mutants. Chapters 5 and 6 explain in detail about the structure solution of dimeric form of PfFabZ, the pH induced conformational flipping of two cis-trans peptide flips that lead to different oligomeric states, and the structural basis for these oligomeric shifts. The mechanism of action of PfFabZ inhibitors NAS-21 and NAS-91 are also discussed in detail.
Intrigued by the hot dog fold of the Fab enzyme, we have analyzed and compared proteins having this fold in their structures. It has been observed that the fold is often associated with fatty acids. However, the sequences, the quaternary structures, substrate specificities and the
reactions that the proteins catalyze are quite diverse. The consensus sequence motifs lining the interface of quaternary association and at active site clearly indicated that the information for different modes of quaternary associations is embedded in the sequences itself. The diversity in function and quaternary association of hot dog fold proteins and their structure-function relationships are discussed in chapter 7.
Malaria affects hundreds of millions of people worldwide causing about two million deaths every year. In spite of the commendable success of the available antimalarials, it has not been possible to contain the disease completely as the protozoan has become resistant to a majority
of frontline drugs. The structural studies presented here should enhance the current biochemical knowledge to develop selective and more potent inhibitors of the pathway and contribute to the ongoing efforts to fight the disease.
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Krause, Robert Gerd Erich. "The detection of two plasmodium falciparum metabolic enzymes using chicken antibodies." Thesis, 2012. http://hdl.handle.net/10413/8522.
Full textAbstract:
Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified.
PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated.
These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered.Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.
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Mtombeni, Nokuhle. "The characterization of the phosphatidyl-inositol-3-kinase in plasmodium falciparum and the effect of selective inhibitors of this enzyme on the parasite." Thesis, 2004. https://hdl.handle.net/10539/25720.
Full textAbstract:
Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine
Johannesburg, 2004Malaria is the most prevalent parasitic disease in the world and the emergence of drug resistant strains of Plasmodium falciparum has made the search for new antimalarial drugs important. Protein kinases play an important role in cellular function and the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in diverse cellular processes such as glucose transport, cell survival and proliferation. A homology based approach identified an open reading frame (ORF) coding for the catalytic region of part of the 6.4 Kb ORF of PFE0765w gene sequence found at plasmoDB. The ORF consisted of 1 758 base pairs which coded for a 586 amino acid protein with a molecular weight of 68.5 KDa. The PfPI3K ORF was amplified from P.falciparum DNA, subcloned into an expression vector and the sequence verified. Analysis of the expressed protein obtained by Western blotting and probing with anti-His monoclonal antibody showed a protein of 68.5 KDa as well as some smaller products.
IT2018
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Krüger, Tim [Verfasser]. "Charakterisierung von Phosphatidylinositol-metabolisierenden Enzymen in Plasmodium falciparum / vorgelegt von Tim Krüger." 2008. http://d-nb.info/989544087/34.
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Yang, Hao. "Role of host haem biosynthetic enzymes in the Plasmodium falciparum erythrocyte-stage." Phd thesis, 2019. http://hdl.handle.net/1885/157107.
Full textAbstract:
Plasmodium falciparum is a parasite that causes severe type of malaria, including cerebral malaria. Its growth and survival in erythrocytes relies on haem, an essential metabolic cofactor participating in various redox reactions of nearly all eukaryotic organisms. Research has therefore focused on interfering with the acquisition of haem by studying three ways parasite can access haem. The first way is through its own de novo haem synthetic pathway (HSP), in which succinyl-CoA and glycine are converted into haem through a series of eight enzymatic steps. The second and third ways are to scavenge either host haem biosynthetic enzymes or haem from haemoglobin degradation, or both, to meet its haem requirements. However, it remains unclear which is the essential way. A recent study has found that host red cells deficient in one of the HSP enzymes, ferrochelatase, gives rise to resistance to malarial infection (in mice) and parasite growth (in human erythropoietic protoporphyric cells), implying that Plasmodium at least requires host FECH enzyme for its growth (Smith, Jerkovic et al. 2015). This project aims to investigate if other host haem biosynthetic enzymes in addition to FECH are required for Plasmodium to infect and propagate in the erythrocyte.
Here we conducted P. falciparum growth assays on human porphyric red blood cells to investigate the parasite requirement for these host haem enzymes. We tested 14 individual AIP patient blood samples; 13 samples showed significant parasite growth inhibitory effect. We also tested the growth of a previously generated P. falciparum HMBS knockout parasite line in normal red blood cells and found the knockout of the PfHmbs had no significant effect on the growth of parasite, which is consistent with a previous study (Sigala, Crowley et al. 2015). However, when cultured in the AIP red cells, the growth of PfHmbs-knockout parasite line was significantly impaired compared to the wild type parental parasite line. This suggests that the lack of PfHmbs may increase the parasite's reliance on the hHMBS. Whole genomic sequencing of the knockout and parental parasite lines was conducted to investigate the hypothesis that the selection and isolation of a viable knockout parasite line occurred through the acquisition of genetic changes that enabled this altered reliance to occur. However, no significant genomic structure changes were consistent with this hypothesis. This work also established an assay to measure human HMBS activity, and used this to screen for inhibitors in a compound library called the Pathogen Box. However none of the approximately 400 compounds inhibited the enzyme. Together, this work identified human HMBS as a novel host enzyme that is required to sustain the parasite growth. Future studies are needed to address the mechanism of enzyme importation by the parasite, and identify small molecule inhibitors of HMBS that may be further developed as novel host-directed antimalarial therapies.