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1.
In vitro selection of high-affinity nucleic acid ligands to parasite target molecules 总被引:2,自引:0,他引:2
The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules. 相似文献
2.
Blood erythrocytes of 25 confirrhed malarial patients infested with P. vivax were analyzed for peroxidation and hemolysis and results compared with 10 uninfected normal control samples. Results indicated significant increase in peroxide formation measured as malondialdehyde, both in presence and absence of H2O2, in parasite infested erythrocytes. These changes induced hemolysis of infected erythrocytes which was increased manifold in presence of H2O2 and could probably be the reason for extensive anemia reported in malaria. 相似文献
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Glutathione S-transferase of the malarial parasite Plasmodium falciparum: characterization of a potential drug target 总被引:7,自引:0,他引:7
Glutathione S-transferases (GSTs), which occur abundantly in most organisms, are essentially involved in the intracellular detoxification of numerous substances including chemotherapeutic agents, and thus play a major role in the development of drug resistance. A gene encoding a protein with sequence identity of up to 37% with known GSTs was identified on chromosome 14 of the malarial parasite Plasmodium falciparum. It was amplified using gametocyte cDNA and expressed in Escherichia coli as a hexahistidyl-tagged protein of 26 kDa subunit size. The homodimeric enzyme (PfGST) was found to catalyse the glutathione (GSH)-dependent modification of 1-chloro-2,4-dinitrobenzene and other typical GST substrates such as o-nitrophenyl acetate, ethacrynic acid, and cumene hydroperoxide. The Km value for GSH was 164+/-20 microM. PfGST was inhibited by cibacron blue (Ki=0.5 microM), S-hexylglutathione (Ki=35 microM), and protoporphyrin IX (Ki=10 microM). Hemin, a most toxic compound for parasitised erythrocytes, was found to be an uncompetitive ligand of PfGST with a Ki of 6.5 microM. Based on the activity of PfGST in extracts of P. falciparum, the enzyme represents 1 to 10% of cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin. Destabilising ligands of GST are thus expected to be synergistic with the antimalarial drug chloroquine, which itself was found to be a very weak inhibitor of PfGST (IC50>200 microM). X-ray quality crystals of PfGST (250x200x50 microm) will serve as starting point for structure-based drug design. 相似文献
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C M Schimandle L Tanigoshi L A Mole I W Sherman 《The Journal of biological chemistry》1985,260(7):4455-4460
Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme. 相似文献
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Lin C Lin K Luong YP Rao BG Wei YY Brennan DL Fulghum JR Hsiao HM Ma S Maxwell JP Cottrell KM Perni RB Gates CA Kwong AD 《The Journal of biological chemistry》2004,279(17):17508-17514
We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061. 相似文献
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The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death. 相似文献
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The human malarial parasite, Plasmodium falciparum, has been found to synthesize heme de novo, despite the accumulation of large quantities of polymeric heme derived from the hemoglobin of the red cell host. The parasite delta-aminolevulinate dehydrase level is significantly lower than that of the host and its inhibition by succinylacetone leads to inhibition of parasite protein synthesis and viability. 相似文献
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H. S. Chawla G. Wenzel 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1987,74(6):841-845
Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive. 相似文献
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Annette N. Chiang Juan-Carlos Valderramos Raghavan Balachandran Raj J. Chovatiya Brian P. Mead Corinne Schneider Samantha L. Bell Michael G. Klein Donna M. Huryn Xiaojiang S. Chen Billy W. Day David A. Fidock Peter Wipf Jeffrey L. Brodsky 《Bioorganic & medicinal chemistry》2009,17(4):1527-1533
Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. 相似文献
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Muthas D Nöteberg D Sabnis YA Hamelink E Vrang L Samuelsson B Karlén A Hallberg A 《Bioorganic & medicinal chemistry》2005,13(18):5371-5390
The increasing resistance of the malarial parasite to antimalarial drugs is a major contributor to the reemergence of the disease and increases the need for new drug targets. The two aspartic proteases, plasmepsins I and II, from Plasmodium falciparum have recently emerged as potential targets. In an effort to inhibit these hemoglobinases, a series of inhibitors encompassing a basic hydroxyethylamine transition state isostere as a central fragment were prepared. The synthesized compounds were varied in the P1' position and exhibited biological activities in the range of 31 to >2000 nM. To try to rationalize the results, molecular docking and 3D-QSAR analysis were used. 相似文献
14.
Spironucleus vortens were cultivated in either an artificial medium at different temperatures, or in medium at various pH conditions or supplemented with different bile concentrations at 25 degrees C. Temperature, pH and bile requirements for the optimal growth of the parasite were determined. Parasites multiplied quickly at 28 and 31 degrees C and reached maximum numbers on Day 4 of cultivation, whereafter they did not survive. At 25 degrees C, parasites survived longer than those at 28 and 31 degrees C with no difference in multiplication rate during the exponential phase. The longest survival period was seen at 22 degrees C, although the growth rate of the parasite was not as high as those at 25 degrees C. At a higher temperature of 37 degrees C, no parasites were observed alive after the second day of cultivation. Optimal pH range for the parasite's growth was 6.5 to 7.5, with the highest cell number at pH 7.5. Parasites survived longest (15 d) at pH 6.0, although the maximum number of cells was lower than those at the optimal pH. Parasites were dead within 24 h at pH levels above 8.5 or below 5.5. All cultures supplemented with either bovine or fish bile yielded numbers of parasites lower than cultures with no bile. In addition, parasite growth was significantly suppressed in medium supplemented with higher concentrations of bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25 degrees C and pH 6.5 to 7.5 without supplementation with bile. 相似文献
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Agnieszka M. Szemiel Andres Merits Richard J. Orton Oscar A. MacLean Rute Maria Pinto Arthur Wickenhagen Gauthier Lieber Matthew L. Turnbull Sainan Wang Wilhelm Furnon Nicolas M. Suarez Daniel Mair Ana da Silva Filipe Brian J. Willett Sam J. Wilson Arvind H. Patel Emma C. Thomson Massimo Palmarini Alain Kohl Meredith E. Stewart 《PLoS pathogens》2021,17(9)
Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance. 相似文献
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Farrow RE Green J Katsimitsoulia Z Taylor WR Holder AA Molloy JE 《Seminars in cell & developmental biology》2011,(9):953-960
Plasmodium falciparum is the most virulent causative agent of malaria in man accounting for 80% of all malarial infections and 90% of the one million annual deaths attributed to malaria. P. falciparum is a unicellular, Apicomplexan parasite, that spends part of its lifecycle in the mosquito and part in man and it has evolved a special form of motility that enables it to burrow into animal cells, a process termed “host cell invasion”. The acute, life threatening, phase of malarial infection arises when the merozoite form of the parasite undergoes multiple cycles of red blood cell invasion and rapid proliferation. Here, we discuss the molecular machinery that enables malarial parasites to invade red blood cells and we focus particularly on the ATP-driven acto-myosin motor that powers invasion. 相似文献
17.
Johnson JJ Roberts CW Pope C Roberts F Kirisits MJ Estes R Mui E Krieger T Brown CR Forman J McLeod R 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(2):966-973
Resistance to murine toxoplasmic encephalitis has been precisely and definitively mapped to the L(d) class I gene. Consistent with this, CD8(+) T cells can adoptively transfer resistance to toxoplasmic encephalitis. However, cytotoxic CD8(+) T cells, capable of killing class I-matched, infected target cells, are generated during the course of Toxoplasma gondii infection even in mice lacking the L(d) gene. L(d)-restricted killing could not be demonstrated, and the functional correlate of the L(d) gene has therefore remained elusive. Herein, L(d)-restricted killing of T. gondii-infected target cells is demonstrated for the first time. L(d)-restricted killing is critically dependent on the strain of T. gondii and is observed with all the derivatives of type II strains tested, but not with a type I strain. These results have important implications for vaccine development. 相似文献
18.
Haploid tobacco (Nicotiana sylvestris) plants were inoculated with a yellow strain of tobacco mosaic virus (TMV-Flavum) and then exposed to 500 rads of acute gamma radiation. Leaf strips cultured on callus-inducing medium yielded two types of colonies: 1) yellow, virus-infected and 2) green, apparently healthy. Of the 3210 calli scored, approximately 5% were virus-free, and after regeneration, 0.2% were resistant at the plant stage. Later, adult plants, both TMV-resistant and TMV-susceptible, produced self-fertile, diploid flowers. Both seedling progeny and rooted cuttings from resistant stock plants showed resistance to TMV infection. This resistance was characterized by restricted virus multiplication and movement within the infected plant resulting in a 3–8 week delay in symptom expression.Journal Paper No. 10138 of the Michigan Agricultural Experiment Station.This work was supported in part by U.S.D.A. grant no. 79-59-2261-1-1-351-1.We thank Drs. M. Daub, R. Griesbach, and J. Hunsperger for helpful suggestions.The excellent technical assistance of Ms. Brenda Floyd and Sara Stadt is acknowledged. 相似文献
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In vitro assays for the study o f erythrocyte invasion by merozoites are available for several primate and rodent malarial species. These assays are essential means by which potential anti-merozoite vaccine candidates are identified. John Dalton, John McNally and Susan O'Donovan describe the various types of invasion assays that are in current use, outline the procedures for performing these assays and add some pointers on interpretation of data derived from them. 相似文献