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Musyoka, Thommas, and Özlem Tastan Bishop. "South African Abietane Diterpenoids and Their Analogs as Potential Antimalarials: Novel Insights from Hybrid Computational Approaches." Molecules 24, no. 22 (November 7, 2019): 4036. http://dx.doi.org/10.3390/molecules24224036.
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The hemoglobin degradation process in Plasmodium parasites is vital for nutrient acquisition required for their growth and proliferation. In P. falciparum, falcipains (FP-2 and FP-3) are the major hemoglobinases, and remain attractive antimalarial drug targets. Other Plasmodium species also possess highly homologous proteins to FP-2 and FP-3. Although several inhibitors have been designed against these proteins, none has been commercialized due to associated toxicity on human cathepsins (Cat-K, Cat-L and Cat-S). Despite the two enzyme groups sharing a common structural fold and catalytic mechanism, distinct active site variations have been identified, and can be exploited for drug development. Here, we utilize in silico approaches to screen 628 compounds from the South African natural sources to identify potential hits that can selectively inhibit the plasmodial proteases. Using docking studies, seven abietane diterpenoids, binding strongly to the plasmodial proteases, and three additional analogs from PubChem were identified. Important residues involved in ligand stabilization were identified for all potential hits through binding pose analysis and their energetic contribution determined by binding free energy calculations. The identified compounds present important scaffolds that could be further developed as plasmodial protease inhibitors. Previous laboratory assays showed the effect of the seven diterpenoids as antimalarials. Here, for the first time, we demonstrate that their possible mechanism of action could be by interacting with falcipains and their plasmodial homologs. Dynamic residue network (DRN) analysis on the plasmodial proteases identified functionally important residues, including a region with high betweenness centrality, which had previously been proposed as a potential allosteric site in FP-2.
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Berger, Louise C., Judith Wilson, Pamela Wood, and Bradley J. Berger. "Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa." Journal of Bacteriology 183, no. 15 (August 1, 2001): 4421–34. http://dx.doi.org/10.1128/jb.183.15.4421-4434.2001.
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ABSTRACT Aspartate aminotransferases have been cloned and expressed fromCrithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, andPlasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549–561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with aKi of 27 μM.
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Eschbach, Marie-Luise, Ingrid B. Müller, Tim-Wolf Gilberger, Rolf D. Walter, and Carsten Wrenger. "The human malaria parasite Plasmodium falciparum expresses an atypical N-terminally extended pyrophosphokinase with specificity for thiamine." Biological Chemistry 387, no. 12 (December 1, 2006): 1583–91. http://dx.doi.org/10.1515/bc.2006.197.
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Abstract Vitamin B1 is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum, are capable of synthesising vitamin B1 de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B1 vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min-1 mg-1 protein and specificity for thiamine with a K m value of 73 μM, while thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of PfTPK with a GFP marker visualised its cytosolic localisation.
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Lande, Duc Hoàng, Abed Nasereddin, Arne Alder, Tim W. Gilberger, Ron Dzikowski, Johann Grünefeld, and Conrad Kunick. "Synthesis and Antiplasmodial Activity of Bisindolylcyclobutenediones." Molecules 26, no. 16 (August 5, 2021): 4739. http://dx.doi.org/10.3390/molecules26164739.
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Malaria is one of the most dangerous infectious diseases. Because the causative Plasmodium parasites have developed resistances against virtually all established antimalarial drugs, novel antiplasmodial agents are required. In order to target plasmodial kinases, novel N-unsubstituted bisindolylcyclobutenediones were designed as analogs to the kinase inhibitory bisindolylmaleimides. Molecular docking experiments produced favorable poses of the unsubstituted bisindolylcyclobutenedione in the ATP binding pocket of various plasmodial protein kinases. The synthesis of the title compounds was accomplished by sequential Friedel-Crafts acylation procedures. In vitro screening of the new compounds against transgenic NF54-luc P. falciparum parasites revealed a set of derivatives with submicromolar activity, of which some displayed a reasonable selectivity profile against a human cell line. Although the molecular docking studies suggested the plasmodial protein kinase PfGSK-3 as the putative biological target, the title compounds failed to inhibit the isolated enzyme in vitro. As selective submicromolar antiplasmodial agents, the N-unsubstituted bisindolylcyclobutenediones are promising starting structures in the search for antimalarial drugs, albeit for a rational development, the biological target addressed by these compounds has yet to be identified.
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Dieckmann, A., and A. Jung. "The mechanism of pyrimethamine resistance in Plasmodium falciparum." Parasitology 93, no. 2 (October 1986): 275–78. http://dx.doi.org/10.1017/s0031182000051441.
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SUMMARYThe uptake of radioactive pyrimethamine by a sensitive and a resistant strain of Plasmodium falciparum, the metabolic fate of pyrimethamine inside these parasites and the kinetic properties of dihydrofolate reductase (DHFR) from both strains have been studied. Uptake of the drug was identical in both strains and no metabolite of pyrimethamine was found in either strain. DHFR from the resistant strain was 300 times less sensitive to inhibition by pyrimethamine than the enzyme from the sensitive strain, while the Michaelis constant for dihydrofolate remained unchanged and inhibition was competitive in both cases. Altered properties of plasmodial DHFR are apparently the only mechanism responsible for pyrimethamine resistance in the strain of Plasmodium falciparum studied.
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Janoff, A., W. J. Roth, S. Sinha, and J. W. Barnwell. "Degradation of plasmodial antigens by human neutrophil elastase." Journal of Immunology 141, no. 4 (August 15, 1988): 1332–40. http://dx.doi.org/10.4049/jimmunol.141.4.1332.
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Abstract Human neutrophil elastase (HNE) has been well-studied with respect to its role in pathologic states, but less is known about the physiologic functions of this important granulocyte enzyme. In the present study, we show that HNE can degrade the major circumsporozoite protein of the infective (sporozoite) stage of Plasmodium vivax malaria, and that this enzyme can also interfere with the cytoadherence of human E infected with Plasmodium falciparum (strain K+ FMG-FCR3) (IE). Cytoadherence reactions are not only blocked by treatment of IE with as little as 10 fg HNE/IE, but already adherent IE are also removed by the enzyme. Normal E surface Ag are not extensively destroyed by these doses of HNE. This suggests that the effect of HNE on cytoadherence is selective and probably due to degradation of the malarial Ag exported to the IE surface and responsible for the formation of "recognition knobs" upon which the cytoadherence reaction depends. This conclusion, in turn, was supported by the results of Western blot analysis showing that HNE degrades a high m.w. Ag found exclusively in membrane extracts of IE. Our results suggest that one physiologic role of HNE may be degradation of parasitic antigens during host defense against malaria.
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TASDEMIR, D., N. GUNER, R. PEROZZO, R. BRUN, A. DONMEZ, I. CALIS, and P. RUEDI. "Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of roots." Phytochemistry 66, no. 3 (February 2005): 355–62. http://dx.doi.org/10.1016/j.phytochem.2004.11.013.
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YEO, Hye-Jeong, Marie-Pierre LARVOR, Marie-Laure ANCELIN, and Henri J. VIAL. "Plasmodium falciparum CTP:phosphocholine cytidylyltransferase expressed in Escherichia coli: purification, characterization and lipid regulation." Biochemical Journal 324, no. 3 (June 15, 1997): 903–10. http://dx.doi.org/10.1042/bj3240903.
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The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) has been isolated from an overexpressing strain of Escherichia coli. The plasmid pETPfCCT mediated the overexpression of the full-length polypeptide directly. The recombinant protein corresponded to 6–9% of the total cellular proteins and was found essentially in the insoluble membrane fraction. Urea at 6 M was used to solubilize the recombinant protein from the insoluble fraction. The CCT activity was restored upon the removal of urea, and the protein was subsequently purified to homogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, including in vitro substrate specificity and enzymic characterization, were assessed. The lipid regulation of the recombinant plasmodial CCT activity was characterized for the first time. The Km values were 0.49±0.03 mM (mean±S.E.M.) for phosphocholine and 10.9±0.5 mM for CTP in the presence of lipid activators (oleic acid/egg phosphatidylcholine vesicles), whereas the Km values were 0.66±0.07 mM for phosphocholine and 28.9±0.8 mM for CTP in the absence of lipid activators. The PfCCT activity was stimulated to the same extent in response to egg phosphatidylcholine vesicles containing anionic lipids, such as oleic acid, cardiolipin and phosphatidylglycerol, and was insensitive or slightly sensitive to PC vesicles containing neutral lipids, such as diacylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme activity by oleic acid was antagonized by the cationic aminolipid sphingosine. These lipid-dependence properties place the parasite enzyme intermediately between the mammalian enzymes and the yeast enzyme.
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Ullah, Najeeb, Hina Andaleeb, Celestin Nzanzu Mudogo, Sven Falke, Christian Betzel, and Carsten Wrenger. "Solution Structures and Dynamic Assembly of the 24-Meric Plasmodial Pdx1–Pdx2 Complex." International Journal of Molecular Sciences 21, no. 17 (August 19, 2020): 5971. http://dx.doi.org/10.3390/ijms21175971.
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Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host’s metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1–Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.
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SINGH, Ajay, Bhaskar R. SHENAI, Youngchool CHOE, Jiri GUT, Puran S. SIJWALI, Charles S. CRAIK, and Philip J. ROSENTHAL. "Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites." Biochemical Journal 368, no. 1 (November 15, 2002): 273–81. http://dx.doi.org/10.1042/bj20020753.
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Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity (kcat/Km increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
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Zidovetzki, R., I. W. Sherman, J. Prudhomme, and J. Crawford. "Inhibition ofPlasmodium falciparumlysophospholipase by anti-malarial drugs and sulphydryl reagents." Parasitology 108, no. 3 (April 1994): 249–55. http://dx.doi.org/10.1017/s0031182000076095.
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SUMMARYThe activity of lysophospholipase of human erythrocytes increased by about 3 orders of magnitude upon infection withPlasmodium falciparum. The apparentKmfor hydrolysis of lysophosphatidylcholine by this enzyme was 50 ± 7μM and the apparentVmax6·8±0·6 nmol/h × 106cells. The activity was Ca2+independent and had a broad pH maximum at pH 8. The enzyme was insensitive to such anti-malarials as mefloquine and arteether and was only weakly inhibited by chloroquine, with a 50% inhibition concentration (IC50) of 70 mM. The anti-malarials quinine and quinacrine were more efficient inhibitors, with IC50s of 2·6 mM and 0·7 mM, respectively. The sulphydryl agentsp–hydroxymercuribenzoate (pHMB) and thimerosal were considerably more potent, inhibiting the plasmodial lysophospholipase with IC50s of 18 μM and 10 μM, respectively. When present at 10 μM prior to invasion, both pHMB and thimerosal arrested the growth and reinvasion capacity ofP. falciparumin culture. In a synchronizedP. falciparumculture the continuous presence of 5 μM thimerosal dramatically decreased total parasitaemia and, within 4 days, totally abolished the capacity of the surviving parasites to reinvade. Thus the plasmodial lysophospholipase may represent a potential new target for anti-malarial chemotherapy.
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Roth, E. Jr, N. Ogasawara, and S. Schulman. "The deamination of adenosine and adenosine monophosphate in Plasmodium falciparum-infected human erythrocytes: in vitro use of 2'deoxycoformycin and AMP deaminase-deficient red cells." Blood 74, no. 3 (August 15, 1989): 1121–25. http://dx.doi.org/10.1182/blood.v74.3.1121.1121.
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Abstract The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2′- deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2′-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high- performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.
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Roth, E. Jr, N. Ogasawara, and S. Schulman. "The deamination of adenosine and adenosine monophosphate in Plasmodium falciparum-infected human erythrocytes: in vitro use of 2'deoxycoformycin and AMP deaminase-deficient red cells." Blood 74, no. 3 (August 15, 1989): 1121–25. http://dx.doi.org/10.1182/blood.v74.3.1121.bloodjournal7431121.
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The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2′- deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2′-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high- performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.
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Kaushal, Deep C., and Nuzhat A. Kaushal. "DIAGNOSIS OF MALARIA BY DETECTION OF PLASMODIAL LACTATE DEHYDROGENASE WITH AN IMMUNODOT ENZYME ASSAY." Immunological Investigations 31, no. 2 (January 2002): 93–106. http://dx.doi.org/10.1081/imm-120004801.
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Schweda, Sandra I., Arne Alder, Tim Gilberger, and Conrad Kunick. "4-Arylthieno[2,3-b]pyridine-2-carboxamides Are a New Class of Antiplasmodial Agents." Molecules 25, no. 14 (July 13, 2020): 3187. http://dx.doi.org/10.3390/molecules25143187.
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Malaria causes hundreds of thousands of deaths every year, making it one of the most dangerous infectious diseases worldwide. Because the pathogens have developed resistance against most of the established anti-malarial drugs, new antiplasmodial agents are urgently needed. In analogy to similar antiplasmodial ketones, 4-arylthieno[2,3-b]pyridine-2-carboxamides were synthesized by Thorpe-Ziegler reactions. In contrast to the related ketones, these carboxamides are only weak inhibitors of the plasmodial enzyme PfGSK-3 but the compounds nevertheless show strong antiparasitic activity. The most potent representatives inhibit the pathogens with IC50 values in the two-digit nanomolar range and exhibit high selectivity indices (>100).
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Arnò, Barbara, Ilda D’Annessa, Cinzia Tesauro, Laura Zuccaro, Alessio Ottaviani, Birgitta Knudsen, Paola Fiorani, and Alessandro Desideri. "Replacement of the Human Topoisomerase Linker Domain with the Plasmodial Counterpart Renders the Enzyme Camptothecin Resistant." PLoS ONE 8, no. 7 (July 2, 2013): e68404. http://dx.doi.org/10.1371/journal.pone.0068404.
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Ajima, Ukpe, Johnson Ogoda Onah, and Noel Nenman Wannang. "Synthesis, Characterization and Biological Evaluation of Benzimidazole - Dihydroartemisinin Hybrids as Potential Dual Acting Antimalarial Agents." Mediterranean Journal of Chemistry 9, no. 1 (August 22, 2019): 52–64. http://dx.doi.org/10.13171/mjc91190822625ua.
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Malaria is a parasitic disease caused by various species of the Plasmodium parasite. In 2016, there were about 216 million cases resulting in 445,000 deaths, with sub-saharan Africa bearing the heaviest burden of the disease. The currently recommended treatment for malaria are combination therapies containing Artemisinin (ACT’s). However, the effectiveness of the Artemisinins is being compromised by the emergence of resistance to the drug and this amplifies the need for new antimalarial drugs. The Benzimidazole scaffold is one of the privileged structures in medicinal chemistry and is associated with a number of biological activities including antimalarial activity which may be through inhibition of the Plasmodial plasmepsin II enzyme. The present study utilizes the concept of molecular hybridization to synthesize hybrid compounds that contain two pharmacophores, acting through two distinct mechanisms. The aim is to improve efficacy and possibly prevent or slow down the emergence of parasite resistance. To confirm their structures, the conjugates were purified by chromatography and characterized using Nuclear Magnetic Resonance (NMR), Mass spectrometry and Infra-red spectroscopy. Antimalarial activities of the hybrids were evaluated in-vitro against the 3D7 strain of Plasmodium falciparum using the parasite Lactate dehydrogenase assay. The hybrids were successfully synthesized with yields ranging from 63.48 percent to 67.60 percent and were all active against the parasite. The Mebendazole conjugate of dihydroartemisinin showed the highest activity with IC50 of 6.861 nM and 6.967 nM for the 5-Benzimidazolecarboxylic acid conjugate of dihydroartemisinin. All the compounds showed statistically significant (p < 0.05) increase in activity as compared to Dihydroartemisinin and Chloroquine alone. These hybrid compounds with improved physicochemical and pharmacological properties may serve as templates for the development of a new class of anitmalarial drugs, which possess advantages over existing drugs in terms of effectiveness and also the ability to overcome the problem of resistance during malaria chemotherapy.
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Kırmızıbekmez, Hasan, Ihsan Çalıs, Remo Perozzo, Reto Brun, Ali A. Dönmez, Anthony Linden, Peter Rüedi, and Deniz Tasdemir. "Inhibiting Activities of the Secondary Metabolites ofPhlomis brunneogaleataagainst Parasitic Protozoa and Plasmodial Enoyl-ACP Reductase, a Crucial Enzyme in Fatty Acid Biosynthesis." Planta Medica 70, no. 8 (August 2004): 711–17. http://dx.doi.org/10.1055/s-2004-827200.
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Pareek, Vidhi, Moumita Samanta, Niranjan V. Joshi, Hemalatha Balaram, Mathur R. N. Murthy, and Padmanabhan Balaram. "Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme." ChemBioChem 17, no. 7 (February 29, 2016): 620–29. http://dx.doi.org/10.1002/cbic.201500532.
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Lunev, Sergey, Soraya S. Bosch, Fernando de Assis Batista, Carsten Wrenger, and Matthew R. Groves. "Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum." Acta Crystallographica Section F Structural Biology Communications 72, no. 7 (June 22, 2016): 523–33. http://dx.doi.org/10.1107/s2053230x16008475.
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Thede novopyrimidine-biosynthesis pathway ofPlasmodium falciparumis a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition ofde novopyrimidine biosynthesisviadihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed toN-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure ofPfATC is reported. The space group of thePfATC crystals was determined to be monoclinicP21, with unit-cell parametersa= 87.0,b= 103.8,c= 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presentedPfATC model shares a high degree of homology with the catalytic domain ofEscherichia coliATC. There is as yet no evidence of the existence of a regulatory domain inPfATC. Similarly toE. coliATC,PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.
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Pelleau, Stéphane, Sylvie Diop, Méry Dia Badiane, Joana Vitte, Pierre Beguin, Farida Nato, Bernard M. Diop, Pierre Bongrand, Daniel Parzy, and Ronan Jambou. "Enhanced Basophil Reactivities during Severe Malaria and Their Relationship with the Plasmodium falciparum Histamine-Releasing Factor Translationally Controlled Tumor Protein." Infection and Immunity 80, no. 8 (July 2, 2012): 2963–70. http://dx.doi.org/10.1128/iai.00072-12.
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ABSTRACTRecent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derivedPlasmodium falciparumhistamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruitedP. falciparum-infected patients in Senegal with mild malaria (MM;n= 19) or severe malaria (SM;n= 9) symptoms and healthy controls (HC;n= 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measuredex vivousing fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.
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Ravindra, Gudihal, and Padmanabhan Balaram. "Plasmodium falciparum triosephosphate isomerase: New insights into an old enzyme." Pure and Applied Chemistry 77, no. 1 (January 1, 2005): 281–89. http://dx.doi.org/10.1351/pac200577010281.
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Triosephosphate isomerase (TIM), a central enzyme in the glycolytic pathway, has been the subject of extensive structural and mechanistic investigations over the past 30 years. The TIM barrel is the prototype of the (β/α)8 barrel fold, which is one of the most extensively used structural motifs in enzymes. Mechanistic studies on TIM from a variety of sources have emphasized the importance of loop 6 dynamics for enzyme activity. Several conserved residues in TIM have been investigated by extensive site-directed mutagenesis of the enzyme from yeast, chicken, and trypanosoma. The cloning and sequencing of the TIM gene from the malarial parasite Plasmodium falciparum in 1993 revealed the unexpected mutation of a hitherto conserved residue serine (S96) to phenylalanine (F96). Subsequent results from the genome sequencing programs of Plasmodium falciparum, Plasmodium vivax, and Plasmodium yoelii confirmed the presence of the S96F mutation in malarial parasites. The crystal structure of PfTIM and several inhibitor complexes, including a high-resolution (1.1 Å) structure of the PfTIM 2-phosphoglycerate complex, revealed that loop 6 had a propensity to remain open, even in several ligand bound structures. Furthermore, both open and closed forms could be characterized for the same complex. Since glycolysis is the primary source of ATP for the malarial parasite during the intraerythrocytic stage, glycolytic enzymes present themselves as potential targets for inhibitors. Two distinct approaches have been explored. The use of dimer interface peptides, which interfere with assembly, has proved promising. Inactivation of the enzyme by modification of a cysteine (C13) residue, which lies close to the active site residue, lysine (K12) is another potential strategy. The differential reactivity, of the four-cysteine residues, at positions 13, 126, 196, and 217 in each subunit has been established using electrospray ionization mass spectrometry. Studies of single tryptophan mutants (W11F and W168F) of PfTIM provide a probe to study folding, stability, and inhibitor interactions.
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Pornthanakasem, Wichai, Pinpunya Riangrungroj, Penchit Chitnumsub, Wanwipa Ittarat, Darin Kongkasuriyachai, Chairat Uthaipibull, Yongyuth Yuthavong, and Ubolsree Leartsakulpanich. "Role of Plasmodium vivax Dihydropteroate Synthase Polymorphisms in Sulfa Drug Resistance." Antimicrobial Agents and Chemotherapy 60, no. 8 (May 9, 2016): 4453–63. http://dx.doi.org/10.1128/aac.01835-15.
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ABSTRACTDihydropteroate synthase (DHPS) is a known sulfa drug target in malaria treatment, existing as a bifunctional enzyme together with hydroxymethyldihydropterin pyrophosphokinase (HPPK). Polymorphisms in key residues ofPlasmodium falciparumDHPS (PfDHPS) have been characterized and linked to sulfa drug resistance in malaria. Genetic sequencing ofP. vivaxdhps(Pvdhps) from clinical isolates has shown several polymorphisms at the positions equivalent to those in thePfdhpsgenes conferring sulfa drug resistance, suggesting a mechanism for sulfa drug resistance inP. vivaxsimilar to that seen inP. falciparum. To characterize the role of polymorphisms in thePvDHPS in sulfa drug resistance, various mutants of recombinantPvHPPK-DHPS enzymes were expressed and characterized. Moreover, due to the lack of a continuousin vitroculture system forP. vivaxparasites, a surrogateP. bergheimodel expressingPvhppk-dhpsgenes was established to demonstrate the relationship between sequence polymorphisms and sulfa drug susceptibility and to test the activities ofPvDHPS inhibitors on the transgenic parasites. Both enzyme activity and transgenic parasite growth were sensitive to sulfadoxine to different degrees, depending on the number of mutations that accumulated in DHPS.Kivalues and 50% effective doses were higher for mutantPvDHPS enzymes than the wild-type enzymes. Altogether, the study provides the first evidence of sulfa drug resistance at the molecular level inP. vivax. Furthermore, the enzyme inhibition assay and thein vivoscreening system can be useful tools for screening new compounds for their activities againstPvDHPS.
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VARADHARAJAN, Sundaramurthy, B. K. Chandrashekar SAGAR, Pundi N. RANGARAJAN, and Govindarajan PADMANABAN. "Localization of ferrochelatase in Plasmodium falciparum." Biochemical Journal 384, no. 2 (November 23, 2004): 429–36. http://dx.doi.org/10.1042/bj20040952.
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Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.
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Chen, Xinlu, Tobias G. Köllner, Wangdan Xiong, Guo Wei, and Feng Chen. "Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum." Beilstein Journal of Organic Chemistry 15 (November 28, 2019): 2872–80. http://dx.doi.org/10.3762/bjoc.15.281.
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Terpene synthases (TPSs) are pivotal enzymes for the production of diverse terpenes, including monoterpenes, sesquiterpenes, and diterpenes. In our recent studies, dictyostelid social amoebae, also known as cellular slime molds, were found to contain TPS genes for making volatile terpenes. For comparison, here we investigated Physarum polycephalum, a plasmodial slime mold also known as acellular amoeba. Plasmodia of P. polycephalum grown on agar plates were found to release a mixture of volatile terpenoids consisting of four major sesquiterpenes (α-muurolene, (E)-β-caryophyllene, two unidentified sesquiterpenoids) and the monoterpene linalool. There were no qualitative differences in terpenoid composition at two stages of young plasmodia. To understand terpene biosynthesis, we analyzed the transcriptome and genome sequences of P. polycephalum and identified four TPS genes designated PpolyTPS1–PpolyTPS4. They share 28–73% of sequence identities. Full-length cDNAs for the four TPS genes were cloned and expressed in Escherichia coli to produce recombinant proteins, which were tested for sesquiterpene synthase and monoterpene synthase activities. While neither PpolyTPS2 nor PpolyTPS3 was active, PpolyTPS1 and PpolyTPS4 were able to produce sesquiterpenes and monoterpenes from the respective substrates farnesyl diphosphate and geranyl diphosphate. By comparing the volatile profile of P. polycephalum plasmodia and the in vitro products of PpolyTPS1 and PpolyTPS4, it was concluded that most sesquiterpenoids emitted from P. polycephalum were attributed to PpolyTPS4. Phylogenetic analysis placed the four PpolyTPSs genes into two groups: PpolyTPS1 and PpolyTPS4 being one group that was clustered with the TPSs from the dictyostelid social amoeba and PpolyTPS2 and PpolyTPS3 being the other group that showed closer relatedness to bacterial TPSs. The biological role of the volatile terpenoids produced by the plasmodia of P. polycephalum is discussed.
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Tasdemir, Deniz, David Sanabria, Ina L. Lauinger, Alice Tarun, Rob Herman, Remo Perozzo, Mire Zloh, Stefan H. Kappe, Reto Brun, and Néstor M. Carballeira. "2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests erythrocytic and liver stage Plasmodium infections." Bioorganic & Medicinal Chemistry 18, no. 21 (November 2010): 7475–85. http://dx.doi.org/10.1016/j.bmc.2010.08.055.
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NA, Byoung-Kuk, Bhaskar R. SHENAI, Puran S. SIJWALI, Youngchool CHOE, Kailash C. PANDEY, Ajay SINGH, Charles S. CRAIK, and Philip J. ROSENTHAL. "Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax." Biochemical Journal 378, no. 2 (March 1, 2004): 529–38. http://dx.doi.org/10.1042/bj20031487.
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Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
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Kaihena, Martha, Maria Nindatu, and Abdul Mahid Ukratalo. "Methanol Extract Alstonia scholaris L. R. Br as Hepatoprotective Mice (Mus musculus) Infected with Plasmodium berghei ANKA Strains." Jurnal Penelitian Pendidikan IPA 9, no. 8 (August 25, 2023): 6076–83. http://dx.doi.org/10.29303/jppipa.v9i8.4834.
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Alstonia scholaris L. R. Br is one of the traditional plants that contain natural antioxidant compounds which are thought to be able to repair damage to the liver cells of mice (Mus musculus) infected with Plasmodium berghei strain ANKA. This study aimed to determine the effect of methanol extract of Alstonia scholaris L. R. Br stem bark on levels of SGPT enzymes and liver cells of mice (Mus musculus) infected with Plasmodium berghei strain ANKA. Mice with a body weight of 20-30 g were infected with Plasmodium berghei as much as 0.1 ml per head and left until the percentage of parasitemia reached 1-5%. Then mice (Mus musculus) were given methanol extract of Alstonia scholaris L. R. Br stem bark at doses of 1, 10, 100, and 200 mg/kg BW for 4 consecutive days. After that, surgery was performed to take blood to observe SGPT enzyme levels and mice liver cells to be prepared with Hematoxylin Eosin (HE) staining. The results of ANOVA showed that the methanol extract of Alstonia scholaris L. R. Br stems bark doses of 1 mg/kg BW, 10 mg/kg BW, 100 mg/kg BW and 200 mg/kg BW could reduce SGPT enzyme levels and repair damage to the liver cells of mice infected with Plasmodium berghei ANKA strains
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Zerez, CR, EF Jr Roth, S. Schulman, and KR Tanaka. "Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes." Blood 75, no. 8 (April 15, 1990): 1705–10. http://dx.doi.org/10.1182/blood.v75.8.1705.1705.
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Abstract Plasmodium falciparum-infected red blood cells (RBCs) are characterized by increases in the activity of glycolytic enzymes. Because nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are cofactors in the reactions of glycolysis and pentose phosphate shunt, we have examined NAD and NADP content in P. falciparum-infected RBCs. Although NADP content was not significantly altered, NAD content was increased approximately 10-fold in infected RBCs (66% parasitemia) compared with uninfected control RBCs. To determine the mechanism for the increase in NAD content, we examined the activity of several NAD biosynthetic enzymes. It is known that normal human RBCs make NAD exclusively from nicotinic acid and lack the capacity to make NAD from nicotinamide. We demonstrate that infected RBCs have readily detectable nicotinamide phosphoribosyltransferase (NPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinamide, and abundant nicotinamide deamidase, the enzyme that converts nicotinamide to nicotinic acid, thereby indicating that infected RBCs can make NAD from nicotinamide. In addition, infected RBCs have a threefold increase in nicotinic acid phosphoribosyltransferase (NAPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinic acid. Thus, the increase in NAD content in P falciparum-infected RBCs appears to be mediated by increases in NAD synthesis from both nicotinic acid and nicotinamide.
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Zerez, CR, EF Jr Roth, S. Schulman, and KR Tanaka. "Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes." Blood 75, no. 8 (April 15, 1990): 1705–10. http://dx.doi.org/10.1182/blood.v75.8.1705.bloodjournal7581705.
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Plasmodium falciparum-infected red blood cells (RBCs) are characterized by increases in the activity of glycolytic enzymes. Because nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are cofactors in the reactions of glycolysis and pentose phosphate shunt, we have examined NAD and NADP content in P. falciparum-infected RBCs. Although NADP content was not significantly altered, NAD content was increased approximately 10-fold in infected RBCs (66% parasitemia) compared with uninfected control RBCs. To determine the mechanism for the increase in NAD content, we examined the activity of several NAD biosynthetic enzymes. It is known that normal human RBCs make NAD exclusively from nicotinic acid and lack the capacity to make NAD from nicotinamide. We demonstrate that infected RBCs have readily detectable nicotinamide phosphoribosyltransferase (NPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinamide, and abundant nicotinamide deamidase, the enzyme that converts nicotinamide to nicotinic acid, thereby indicating that infected RBCs can make NAD from nicotinamide. In addition, infected RBCs have a threefold increase in nicotinic acid phosphoribosyltransferase (NAPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinic acid. Thus, the increase in NAD content in P falciparum-infected RBCs appears to be mediated by increases in NAD synthesis from both nicotinic acid and nicotinamide.
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Du, Yu, Jolyn E. Gisselberg, Jacob D. Johnson, Patricia J. Lee, Sean T. Prigge, and Brian O. Bachmann. "Lactococcus lactis fabH, Encoding β-Ketoacyl-Acyl Carrier Protein Synthase, Can Be Functionally Replaced by the Plasmodium falciparum Congener." Applied and Environmental Microbiology 76, no. 12 (April 23, 2010): 3959–66. http://dx.doi.org/10.1128/aem.00170-10.
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ABSTRACT Plasmodium falciparum, in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. β-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in P. falciparum: the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid Lactococcus lactis strain that translates pfKASIII instead of L. lactis f abH to initiate fatty acid biosynthesis. The L. lactis expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in L. lactis. Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the L. lactis strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use L. lactis ACP as substrate and perform near-normal function in L. lactis cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.
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Roth, EF Jr, MC Calvin, I. Max-Audit, J. Rosa, and R. Rosa. "The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites." Blood 72, no. 6 (December 1, 1988): 1922–25. http://dx.doi.org/10.1182/blood.v72.6.1922.1922.
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Abstract Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6- phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.
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Roth, EF Jr, MC Calvin, I. Max-Audit, J. Rosa, and R. Rosa. "The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites." Blood 72, no. 6 (December 1, 1988): 1922–25. http://dx.doi.org/10.1182/blood.v72.6.1922.bloodjournal7261922.
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Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6- phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.
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Kadian, Kavita, Yash Gupta, Harsh Vardhan Singh, Prakasha Kempaiah, and Manmeet Rawat. "Apicoplast Metabolism: Parasite’s Achilles’ Heel." Current Topics in Medicinal Chemistry 18, no. 22 (January 10, 2019): 1987–97. http://dx.doi.org/10.2174/1568026619666181130134742.
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Malaria continues to impinge heavily on mankind, with five continents still under its clasp. Widespread and rapid emergence of drug resistance in the Plasmodium parasite to current therapies accentuate the quest for novel drug targets and antimalarial compounds. Plasmodium parasites, maintain a non-photosynthetic relict organelle known as Apicoplast. Among the four major pathways of Apicoplast, biosynthesis of isoprenoids via Methylerythritol phosphate (MEP) pathway is the only indispensable function of Apicoplast that occurs during different stages of the malaria parasite. Moreover, the human host lacks MEP pathway. MEP pathway is a validated repertoire of novel antimalarial and antibacterial drug targets. Fosmidomycin, an efficacious antimalarial compound against IspC enzyme of MEP pathway is already in clinical trials as a combination drugs. Exploitation of other enzymes of MEP pathway would provide a much-needed impetus to the antimalarial drug discovery programs for the elimination of malaria. We outline the cardinal features of the MEP pathway enzymes and progress made towards the characterization of new inhibitors.
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Bushkin, G. Guy, Daniel M. Ratner, Jike Cui, Sulagna Banerjee, Manoj T. Duraisingh, Cameron V. Jennings, Jeffrey D. Dvorin, et al. "Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii." Eukaryotic Cell 9, no. 2 (September 25, 2009): 228–41. http://dx.doi.org/10.1128/ec.00197-09.
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ABSTRACT We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc3Man5GlcNAc2) and the host 14-sugar precursor (Glc3Man9GlcNAc2) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.
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Carballeira, Néstor M., Angela Gono Bwalya, Maurice Ayamba Itoe, Adriano D. Andricopulo, María Lorena Cordero-Maldonado, Marcel Kaiser, Maria M. Mota, Alexander D. Crawford, Rafael V. C. Guido, and Deniz Tasdemir. "2-Octadecynoic acid as a dual life stage inhibitor of Plasmodium infections and plasmodial FAS-II enzymes." Bioorganic & Medicinal Chemistry Letters 24, no. 17 (September 2014): 4151–57. http://dx.doi.org/10.1016/j.bmcl.2014.07.050.
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KAPOOR, Mili, C. Chandramouli REDDY, M. V. KRISHNASASTRY, Namita SUROLIA, and Avadhesha SUROLIA. "Slow-tight-binding inhibition of enoyl-acyl carrier protein reductase from Plasmodium falciparum by triclosan." Biochemical Journal 381, no. 3 (July 27, 2004): 719–24. http://dx.doi.org/10.1042/bj20031821.
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Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme–inhibitor complex (EI) that isomerizes to a second enzyme–inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49×10−2 and 1×10−4 s−1 respectively. The Ki value for the formation of the EI complex was 53 nM and the overall inhibition constant Ki* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725–733].
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Baldwin, Jeffrey, Carolyn H. Michnoff, Nicholas A. Malmquist, John White, Michael G. Roth, Pradipsinh K. Rathod, and Margaret A. Phillips. "High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase." Journal of Biological Chemistry 280, no. 23 (March 28, 2005): 21847–53. http://dx.doi.org/10.1074/jbc.m501100200.
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Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC50 values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC50 against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.
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Aroonsri, Aiyada, Navaporn Posayapisit, Jindaporn Kongsee, Onsiri Siripan, Danoo Vitsupakorn, Sugunya Utaida, Chairat Uthaipibull, Sumalee Kamchonwongpaisan, and Philip J. Shaw. "Validation of Plasmodium falciparum deoxyhypusine synthase as an antimalarial target." PeerJ 7 (April 17, 2019): e6713. http://dx.doi.org/10.7717/peerj.6713.
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Background Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. Methods Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. Results Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. Discussion Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.
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Sturm, Nicole, Ying Hu, Herbert Zimmermann, Karin Fritz-Wolf, Sergio Wittlin, Stefan Rahlfs, and Katja Becker. "Compounds Structurally Related to Ellagic Acid Show Improved Antiplasmodial Activity." Antimicrobial Agents and Chemotherapy 53, no. 2 (November 17, 2008): 622–30. http://dx.doi.org/10.1128/aac.00544-08.
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ABSTRACT The cancer chemopreventive agent ellagic acid (EA) is a known inhibitor of glutathione S-transferases (GSTs) and possesses antiplasmodial activities in the upper-nanomolar range. In the recent drug development approach, the properties of the active site of Plasmodium falciparum GST were exploited for inhibitor design by introducing one or two additional hydroxyl groups into EA, yielding flavellagic acid (FEA) and coruleoellagic acid (CEA), respectively. Indeed, the inhibition of P. falciparum GST was improved with the increasing hydrophilicity of the planar polyaromatic ring system. Studying the effects of the two compounds on the central redox enzymes of Plasmodium revealed that glutathione reductase and thioredoxin reductase also are inhibited in the lower-micromolar range. Both compounds had strong antiplasmodial activity in the lower-nanomolar range and were particularly effective against chloroquine (CQ)-resistant P. falciparum strains. Neither FEA nor CEA showed cytotoxic effects on human cells. This was supported by negligible changes in transcript levels and enzyme activities of redox enzymes in human A549 cells upon treatment with the compounds. In Plasmodium, however, CEA treatment resulted in a marked downregulation of most antioxidant genes studied and impaired mainly the trophozoite stage of the parasites. In addition, EA, CEA, and FEA were found to strongly inhibit in vitro heme aggregation. In vitro and preliminary in vivo studies indicated that, compared to CQ, CEA is a slowly acting compound and is able to significantly improve the survival of Plasmodium berghei-infected mice. We conclude that FEA and CEA are promising antimalarial compounds that deserve to be studied further.
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Ko, Reamonn, and Masayo Kotaka. "Structural studies of Plasmodium falciparum GTP:AMP phosphotransferase." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C457. http://dx.doi.org/10.1107/s2053273314095424.
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Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010[1]. 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. PfGAK was successfully cloned and expressed in Escherichia Coli. Furthermore, using 2-step chromatography the enzyme was purified and screened for crystallization conditions. PfGAK crystallized into brown hexagonal crystals and diffracted at a 2.9 Å resolution. The apo-structure have been solved and now we are working on determining the structure for PfGAK when bound to its substrate analog GP5A.
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Jortzik, Esther, Boniface M. Mailu, Janina Preuss, Marina Fischer, Lars Bode, Stefan Rahlfs, and Katja Becker. "Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum." Biochemical Journal 436, no. 3 (May 27, 2011): 641–50. http://dx.doi.org/10.1042/bj20110170.
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The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.
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Buchholz, Kathrin, R. Heiner Schirmer, Jana K. Eubel, Monique B. Akoachere, Thomas Dandekar, Katja Becker, and Stephan Gromer. "Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium falciparum." Antimicrobial Agents and Chemotherapy 52, no. 1 (October 29, 2007): 183–91. http://dx.doi.org/10.1128/aac.00773-07.
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ABSTRACT Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M−1 s−1), thioredoxins (k = 8.5 to 26 M−1 s−1), dihydrolipoamide (k = 53 M−1 s−1), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the k cat values ranging from 0.03 s−1 to 10 s−1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.
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Kanchanaphum, Panan, and Jerapan Krungkrai. "Co-expression of human malaria parasite Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5’-monophosphate decarboxylase as enzyme complex in Escherichia coli: a novel strategy for drug development." Asian Biomedicine 4, no. 2 (April 1, 2010): 297–306. http://dx.doi.org/10.2478/abm-2010-0037.
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Abstract Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway. The fifth and sixth enzymes of the pathway form a heterotetrameric complex, containing two molecules each of orotate phosphoribosyltransferase (OPRT) and orotidine 5’-monophosphate decarboxylase (OMPDC). Objective: Define the function of OPRT-OMPDC enzyme complex of P. falciparum by co-expressing the enzymes in Escherichia coli. Methods: The constructed plasmids containing either P. falciparum OPRT or OMPDC were cloned in E. coli by co-transformation. Both genes were co-expressed as OPRT-OMPDC enzyme complex and the complex was purified by chromatographic techniques, including N2+-NTA affinity, Hi Trap Q HP anion-exchange, uridine 5’- monophosphate affinity, and Superose 12 gel-filtration columns. Physical and kinetic properties of the enzyme complex were analyzed for its molecular mass. Results: Co-transformation of PfOPRT and PfOMPDC plasmids in E. coli were achieved with a clone containing DNA ratio of 1:2, respectively. Both plasmids remained stable and were functionally expressed in the E. coli cell for at least 20 weeks. The P. falciparum OPRT-OMPDC enzyme complex were co-expressed and the complex was co-eluted in all chromatographic columns during purification and physical analysis. The molecular mass of the complex was 130 kDa, whereas the PfOPRT and PfOMPDC component were 35.6 and 41.5 kDa, respectively. The enzymatic activities of the complex were competitively inhibited by their products of each enzyme component. Conclusion: P. falciparum OPRT and OMPDC in E. coli as an enzyme complex were co-transformed and functionally co-expressed. These have similar properties to the native enzyme purified directly from P. falciparum, and this character is different from that of the human host organism. The enzyme complex would be suitable as new target to research selective inhibitors as suitable drugs to better control this disease.
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KANDEEL, M., T. ANDO, Y. KITAMURA, M. ABDEL-AZIZ, and Y. KITADE. "Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery." Parasitology 136, no. 1 (January 2009): 11–25. http://dx.doi.org/10.1017/s0031182008005301.
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SUMMARYPlasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.
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Hariharan, Jayashree, Rajendra Rane, Kasirajan Ayyanathan, Philomena, Vidya Prasanna Kumar, Dwarkanath Prahlad, and Santanu Datta. "Mechanism-Based Inhibitors: Development of a High Throughput Coupled Enzyme Assay to Screen for Novel Antimalarials." Journal of Biomolecular Screening 4, no. 4 (August 1999): 187–92. http://dx.doi.org/10.1177/108705719900400406.
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Identifying potent enzyme inhibitors through a robust HTS assay is currently thought to be the most efficient way of searching for lead molecules. We have developed a HTS assay that mimics a crucial step in an essential metabolic pathway, the purine salvage pathway of the malarial parasite Plasmodium falciparum. In this assay we have used purified recombinant enzymes: hypoxanthine guanine phosphoribosyl transferase (HGPRT) and inosine monophosphate dehydrogenase (IMPDH) from the malarial parasite and the human host, respectively. These two enzymes, which work in tandem, are used to set up a coupled assay that is robust enough to meet the stringent criteria of an HTS assay. In the first phase of our screen we seem to have identified novel inhibitors that kill the parasite by inhibiting the salvage pathway of the parasite.
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Brophy, Victoria Hertle, John Vasquez, Richard G. Nelson, John R. Forney, Andre Rosowsky, and Carol Hopkins Sibley. "Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae." Antimicrobial Agents and Chemotherapy 44, no. 4 (April 1, 2000): 1019–28. http://dx.doi.org/10.1128/aac.44.4.1019-1028.2000.
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ABSTRACT There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinantC. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvumDHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.
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Bhunya, Rajabrata, Suman Nandy, and Alpana Seal. "An in silico structural insights into Plasmodium LytB protein and its inhibition." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1791. http://dx.doi.org/10.1107/s2053273314082096.
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In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new antimalarial compounds or inhibitors. Here a systematic in-silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three dimensional (3D) structure of Plasmodium LytB taking E. Coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand dataset containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, 5 were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB inhibitory mechanism which can provide valuable support for the antimalarial inhibitor design in future.
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Hastings, Michele D., Jason D. Maguire, Michael J. Bangs, Peter A. Zimmerman, John C. Reeder, J. Kevin Baird, and Carol Hopkins Sibley. "Novel Plasmodium vivax dhfr Alleles from the Indonesian Archipelago and Papua New Guinea: Association with Pyrimethamine Resistance Determined by a Saccharomyces cerevisiae Expression System." Antimicrobial Agents and Chemotherapy 49, no. 2 (February 2005): 733–40. http://dx.doi.org/10.1128/aac.49.2.733-740.2005.
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ABSTRACT In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.
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Dahalan, Farah Aida, Hasidah Mohd Sidek, Mogana Das Murtey, Mohammed Noor Embi, Jamaiah Ibrahim, Lim Fei Tieng, Nurul Aiezzah Zakaria, and Noraishah Mydin Abdul-Aziz. "Phosphorylated and Nonphosphorylated PfMAP2 Are Localized in the Nucleus, Dependent on the Stage ofPlasmodium falciparumAsexual Maturation." BioMed Research International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/1645097.
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Plasmodium falciparummitogen-activated protein (MAP) kinases, a family of enzymes central to signal transduction processes including inflammatory responses, are a promising target for antimalarial drug development. Our study shows for the first time that theP. falciparumspecific MAP kinase 2 (PfMAP2) is colocalized in the nucleus of all of the asexual erythrocytic stages ofP. falciparumand is particularly elevated in its phosphorylated form. It was also discovered that PfMAP2 is expressed in its highest quantity during the early trophozoite (ring form) stage and significantly reduced in the mature trophozoite and schizont stages. Although the phosphorylated form of the kinase is always more prevalent, its ratio relative to the nonphosphorylated form remained constant irrespective of the parasites’ developmental stage. We have also shown that the TSH motif specifically renders PfMAP2 genetically divergent from the other plasmodial MAP kinase activation sites using Neighbour Joining analysis. Furthermore, TSH motif-specific designed antibody is crucial in determining the location of the expression of the PfMAP2 protein. However, by using immunoelectron microscopy, PPfMAP2 were detected ubiquitously in the parasitized erythrocytes. In summary, PfMAP2 may play a far more important role than previously thought and is a worthy candidate for research as an antimalarial.