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The Journal of Biological Chemistry Sep 2006Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development....
Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.
Topics: Amino Acid Sequence; Animals; Escherichia coli; Humans; Inhibitory Concentration 50; Kinetics; Molecular Sequence Data; Nucleotides; Plasmodium falciparum; Purine-Nucleoside Phosphorylase; Sequence Homology, Amino Acid; Substrate Specificity; Toxoplasma
PubMed: 16829527
DOI: 10.1074/jbc.M602624200 -
European Journal of Biochemistry Nov 1979The occurrence of 5'-methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87 degrees C, is reported. It represents...
The occurrence of 5'-methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87 degrees C, is reported. It represents the first example in prokaryotes of a phosphoryolytic cleavage of the thioether. The reaction products, purified by ion-exchange chromatography, have been identified as 5-methylthioribose-1-phosphate and adenine by several analytical procedures. The enzyme has been purified to homogeneity in 32% yield by using DEAE-cellulose and hydroxyapatite chromatography, gel filtration and isoelectric focusing. The enzyme shows a high degree of thermophilicity, its temperature optimum being at 93 degrees C; furthermore no loss of activity is observable after exposure for 1 h at 100 degrees C. The kinetic data indicate a sequential mechanism of the reaction. The apparent Km values are 0.095 mM for 5'-methylthioadenosine and 6.1 mM for phosphate. The specificity of the reaction is rather strict. Experiments performed with analogues of the substrate, i.e. 5'-methylthioinosine, 5'-dimethylthioadenosine sulfonium salt, 5'-n-butylthioadenosine, 5'-isobutylthioadenosine, 5'-isobutylthioinosine, adenosylhomocysteine, 5'-thioethanoladenosine, adenosine, indicate the relevance of the adenine amino group and the sulfur in thioether form in the binding to the enzyme protein.
Topics: Adenosine; Bacteria; Drug Stability; Hot Temperature; Kinetics; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides
PubMed: 118001
DOI: 10.1111/j.1432-1033.1979.tb19723.x -
Journal of Pharmaceutical and... Sep 2014In the treatment of inflammatory bowel diseases, the use of azathioprine is increasing over the time. It has been demonstrated that the effectiveness of this therapy is...
In the treatment of inflammatory bowel diseases, the use of azathioprine is increasing over the time. It has been demonstrated that the effectiveness of this therapy is modulated by the metabolism of azathioprine, which is mainly exerted by both thiopurine methyl-transferase and inosine triphosphatase enzymes. Several studies reported chromatographic methods to determine the amount of its metabolites in erythrocytes, but there are not reported methods to dose them in peripheral blood mononuclear cells (PBMCs). The development of a method capable to quantify azathioprine nucleoside metabolites in this compartment could give better information on drug penetration and metabolism in the active site. In this work, we validated a new chromatographic method suitable for the monitoring of the two major biologically active ribonucleos(t)ide metabolites of azathioprine in PBMCs: 6-thioguanosine and 6-methyl-mercaptopurine riboside. After PBMCs extraction from blood through separation on density gradient, samples underwent a de-phosphorylation procedure with acid phosphatase (only one aliquot for each sample) and were then treated with a protein precipitation protocol in acetonitrile, followed by UPLC-tandem-mass spectrometry analysis. The calibration curve for each metabolite in PBMC fitted a least squares model (weighed 1/X) from 0.048 to 25ng (r(2)=0.998). Both accuracy and precision parameters fitted FDA guidelines. We tested this method by monitoring the concentrations of each metabolite in PBMC from eight inflammatory bowel diseases affected patients, receiving azathioprine maintenance therapy with optimal results.
Topics: Azathioprine; Chromatography, High Pressure Liquid; Guanosine; Humans; Inflammatory Bowel Diseases; Leukocytes, Mononuclear; Methylthioinosine; Tandem Mass Spectrometry; Thionucleosides
PubMed: 24960235
DOI: 10.1016/j.jpba.2014.05.040 -
The Journal of Biological Chemistry Feb 1981
Topics: Adenine; Cell Division; Feedback; Fibroblasts; Humans; Hypoxanthines; Inosine; Kinetics; Methylthioinosine; Phosphotransferases; Ribose-Phosphate Pyrophosphokinase; Skin
PubMed: 6257688
DOI: No ID Found -
Nucleic Acids Research Aug 2003The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich...
The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm and the contribution of these hypermodified nucleosides to codon-anticodon interactions, as well as translation, are significant, although not fully understood. Herein we described a new chemical synthesis method of the oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N6-alkyladenosines, the precursor oligoribonucleotide carrying 6-methylthiopurine riboside residue was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N6-alkyladenosines the precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. Among the modified oligoribonucleotides of different length and secondary structures, there were several containing naturally occurring modified nucleosides such as: N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), and 2-methylthio-N6-methyladenosine (ms2m6A), as well as several unnaturally modified adenosine derivatives.
Topics: Adenosine; Alkylation; Amines; Methylthioinosine; Oligonucleotides; Oligoribonucleotides; Organophosphorus Compounds; Oxidation-Reduction; Purine Nucleosides; Sulfoxides; Thionucleosides
PubMed: 12888506
DOI: 10.1093/nar/gkg632 -
Molecular Biology of the Cell Jan 1993Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The...
Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.
Topics: 2-Aminopurine; Animals; Cell Line; Methylthioinosine; Myelin Basic Protein; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Nerve Growth Factor; Thioguanine
PubMed: 7680248
DOI: 10.1091/mbc.4.1.71 -
Biochemical Pharmacology Jan 19956-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine...
6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.
Topics: Adenine; Adenosine; Antineoplastic Agents; Cell Division; Cell Survival; Cells, Cultured; Guanosine; Humans; Inosine; Mercaptopurine; Methylthioinosine; Nucleosides; Nucleotides; Purine Nucleosides; Ribonucleosides; T-Lymphocytes; Thioinosine; Thionucleotides
PubMed: 7840782
DOI: 10.1016/0006-2952(94)00387-2 -
Molecules (Basel, Switzerland) Aug 20056-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the...
6-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the beta-D-anomer exclusively. The in vitro antitumor effects of beta-D-MPR and 6-methyl-purine-alpha-D-riboside (alpha-D-MPR) in five human tumor cell lines showed that beta-D-MPR was highly active (IC(50) values ranging from 6 to 34 nM). alpha-D-MPR, although less active than beta-D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83 microM).
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Methylthioinosine; Stereoisomerism
PubMed: 18007371
DOI: 10.3390/10081015 -
Acta Poloniae Pharmaceutica 2000The kinetics of hydrolysis of ribosilo-6-methylmercaptopurine was studied in aqueous solution at 353 K over a pH range of 0.45-12.13. The decomposition was followed by...
The kinetics of hydrolysis of ribosilo-6-methylmercaptopurine was studied in aqueous solution at 353 K over a pH range of 0.45-12.13. The decomposition was followed by HPLC method. The reaction of ribosilo-6-methylmercaptopurine hydrolysis includes: the reaction catalysed by hydrogen ions, that catalysed by hydroxide ions and spontaneous hydrolysis under the effect of water.
Topics: Chromatography, High Pressure Liquid; Drug Stability; Hydrolysis; Indicators and Reagents; Kinetics; Methylthioinosine; Solutions
PubMed: 11126616
DOI: No ID Found -
Nucleosides, Nucleotides & Nucleic Acids Jun 2010Monitoring of thiopurine metabolites is important due to a complex metabolism with large interindividual variation, but the suitability of currently used methods has...
Monitoring of thiopurine metabolites is important due to a complex metabolism with large interindividual variation, but the suitability of currently used methods has been questioned. The drawbacks include poor reproducibility, the inability to differentiate between the different analytes, as well as the use of a nontarget matrix. Further research should be directed toward measuring thiopurine metabolites in mononuclear cells, measuring the different nucleotides specifically, as well as measuring the incorporation of thioguanine into DNA. The studies should not be limited to thioguanosine nucleotides but include methylthioinosine nucleotides as well.
Topics: Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Mercaptopurine; Purines; Thioguanine
PubMed: 20544507
DOI: 10.1080/15257771003741133