Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0024530 (malaria)
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Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.
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PMID:Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. 938 Jul 37

The methemoglobin reductase system plays a vital role in maintaining the equilibrium between hemoglobin and methemoglobin in blood. Exposure of red blood cells to oxidative stress (pathological/physiological) may cause impairment to this equilibrium. We studied the status of erythrocytic methemoglobin and the related reductase system during Plasmodium yoelii nigeriensis infection in mice and P. berghei infection in mastomys. Malaria infection was induced by intraperitoneal inoculation with 10(6) infected erythrocytes. The present investigation revealed a significant decrease in the activity of methemoglobin reductase, with a concomitant rise in methemoglobin content during P. yoelii nigeriensis infection in mice erythrocytes. This was accompanied with a significant increase in reduced glutathione and ascorbate levels. The activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase increased with a progressive rise in parasitemia. However, no methemoglobin or associated reductase activity was detected in normal and P. berghei-infected mastomys. P. berghei infection in mastomys resulted in an increase in the level of reduced glutathione and ascorbate in erythrocytes, and also in the activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase. These results suggest that antioxidants/antioxidant enzymes may prevent or reduce the formation of methemoglobin in the host and thereby protect the host from methemoglobinemia.
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PMID:Changes in rodent-erythrocyte methemoglobin reductase system produced by two malaria parasites, viz. Plasmodium yoelii nigeriensis and Plasmodium berghei. 1143 27

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.
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PMID:Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum. 1146 68

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.
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PMID:Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase. 1179 10

Malaria is one of the most devastating tropical diseases despite the availability of numerous drugs acting against the protozoan parasite Plasmodium in its human host. However, the development of drug resistance renders most of the existing drugs useless. In the malaria parasite the tripeptide glutathione is not only involved in maintaining an adequate intracellular redox environment and protecting the cell against oxidative stress, but it has also been shown that it degrades non-polymerized ferriprotoporphyrin IX (FP IX) and is thus implicated in the development of chloroquine resistance. Glutathione levels in Plasmodium -infected red blood cells are regulated by glutathione synthesis, glutathione reduction and glutathione efflux. Therefore the effects of drugs that interfere with these metabolic processes were studied to establish possible differences in the regulation of the glutathione metabolism of a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodium falciparum. Growth inhibition of P. falciparum 3D7 by D,L-buthionine-( S, R )sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), and by Methylene Blue (MB), an inhibitor of gluta thione reductase (GR), was significantly more pronounced than inhibition of P. falciparum Dd2 growth by these drugs. These results correlate with the higher levels of total glutathione in P. falciparum Dd2. Short-term incubations of Percoll-enriched trophozoite-infected red blood cells in the presence of BSO, MB and N, N (1)-bis(2-chloroethyl)- N -nitrosourea and subsequent determinations of gamma-GCS activities, GR activities and glutathione disulphide efflux revealed that maintenance of intracellular glutathione in P. falciparum Dd2 is mainly dependent on glutathione synthesis whereas in P. falciparum 3D7 it is regulated via GR. Generally, P. falciparum Dd2 appears to be able to sustain its intracellular glutathione more efficiently than P. falciparum 3D7. In agreement with these findings is the differential susceptibility to oxidative stress of both parasite strains elicited by the glucose/glucose oxidase system.
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PMID:Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. 1222 91

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.
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PMID:Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data. 1260 58

Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical importance. They include the human pathogens Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease, together affecting several hundred million people worldwide. The search for new and effective drugs against these pathogens has been boosted during the last years by an unexpected finding. Through molecular and cell biological analysis it was realized that probably most members of this phylum harbor a plastid-like organelle, called the apicoplast, which probably is derived from the engulfment of a red alga in ancient times. Although the apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal "playground" for those interested in drug targets. Recent reports have shown that the rationale of this approach is valid and that new drugs which are urgently needed especially for plasmodial infections, might be developed in the near future based on these targets. Amongst them are three enzymes of the plant-like fatty acid synthesis machinery and enzymes of the non-mevalonat isoprenoid biosynthesis pathway. From their presence in the apicoplast it can be concluded that fatty acid and lipid biosynthesis seems to be a major function of the apicoplast. Another recently described apicoplast enzyme, ferredoxin-NADP(+)-reductase and its redox partner, ferredoxin, points to another interesting organelle-specific biosynthetic pathway, namely [Fe-S] cluster biosynthesis. In the present review, the fundamental aspects of the apicoplast as drug target will be described, together with the specific pathways and their currently known inhibitors. Furthermore, based on the recent findings potentially new targets will be discussed. A short overview of the presently available high-throughput methods for Apicomplexa to evaluate the potency of new inhibitory substances will also be given.
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PMID:Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic apicomplexa. 1276 82

New hope has been gained in the control of the malaria parasite Plasmodium falciparum (pf) with the discovery that the parasite contains a prokaryotic type II fatty-acid synthase (FAS). Since enzymes of this type are absent in humans, they are potential targets for the development of new drugs. The enoyl reductase enzyme (ENR) belonging to this pathway is of particular interest because it has been shown to be inhibited by submicromolar concentrations of the antimicrobial agent triclosan. Here, the development of an efficient overexpression system for pfENR as a fusion protein with maltose-binding protein, its simple one-step purification and cleavage from its fusion protein and crystallization under new conditions with bound NAD(+) cofactor and triclosan are reported. The crystals belong to the space group P2(1), with approximate unit-cell parameters a = 88.2, b = 82.4, c = 94.8 A, beta = 90.77 degrees, and contain a tetramer in the asymmetric unit. Cryocooled crystals (100 K) diffracted to beyond 2.2 A resolution at the Daresbury Synchrotron Radiation Source.
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PMID:Expression, purification and crystallization of the Plasmodium falciparum enoyl reductase. 1283 74

Malaria remains a major disease of mankind, and resistance to existing therapeutics is rapidly emerging. Limited financial investment to develop new therapeutics requires the careful selection of well-defined targets from the causative parasite, Plasmodium falciparum. In these circumstances, protein crystallography can provide valuable structural detail to facilitate both the selection of suitable targets and the development of compounds to provide novel drug candidates. This review summarises the current involvement of crystallographic studies in anti-malarial drug development programmes. Protein crystallography is increasingly central to the exploitation of a number of potential Plasmodial targets. including the aspartic acid proteases (plasmepsins) and cysteine proteases (falcipains) involved in haem degradation within the parasite food vacuole. Lead compounds are being identified from collections previously synthesised against homologous human enzymes. Plasmodium have an unusual dependence on the glycolytic pathway relative to their human hosts, and this is reflected in subtle structural differences identified in the crystal structures of a number of parasite glycolytic enzymes including aldolase and lactate dehydrogenase. Other enzymes from a range of biosynthetic pathways have also been targeted in crystallographic studies. These include dihydrofolate reductase, the target of existing anti-folate therapeutics, and enoyl reductase from the fatty acid biosynthesis pathway which is already the target of effective bacteriocides. Crystal structures of these drug-enzyme complexes not only allow visualisation and improvement of inhibitor-protein contacts, but in the former case have also been used to probe the molecular basis of emerging anti-malarial drug resistance. Crystallography is similarly proving valuable as a tool to facilitate the development of inhibitors of purine salvage, isoprenoid synthesis and utilisation, and protein processing mechanisms.
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PMID:Structure-based approaches to the development of novel anti-malarials. 1501 47

Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast, its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD+ [Suguna, A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283, 224-228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277, 13106-13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.
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PMID:Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum. 1513 52


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