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Molecular Cancer Therapeutics Mar 2011The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into...
The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.
Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Deletion; Humans; Immunoblotting; Mercaptopurine; Neoplasms; Purine-Nucleoside Phosphorylase; Purines; Thioguanine; Thioinosine; Thionucleotides
PubMed: 21282358
DOI: 10.1158/1535-7163.MCT-10-0798 -
The Journal of Biological Chemistry Jan 1984Methylthioinosine (MeSno) is a purine nucleoside analog which is cytotoxic to a number of cultured cell lines including the Reuber H35 hepatoma cells used in the present...
Methylthioinosine (MeSno) is a purine nucleoside analog which is cytotoxic to a number of cultured cell lines including the Reuber H35 hepatoma cells used in the present studies. It has also been observed to cause a rapid profound loss of tyrosine aminotransferase activity in H35 cells well before the onset of any measurable cytotoxicity. The effect is both time and concentration dependent. MeSno does not acutely inhibit synthesis of the enzyme as evidenced by the ability of glucocorticoids or cAMP analogs to induce the enzyme to the same extent in the presence or absence of the drug. The enzyme in extracts of cells treated with the drug is essentially identical with the enzyme from extracts of control cells in terms of thermal stability, immunoprecipitability, and affinities for substrates and cofactor. Addition of MeSno to cell extracts and mixing experiments suggests that the thiopurine does not have any direct effect on enzyme activity. Immunochemical analysis of the rates of synthesis and degradation of the aminotransferase have shown that the enzyme is degraded approximately 3-4 times more rapidly in cells treated with the drug than in control cells. At the same time there is no inhibition of the rate of synthesis of the enzyme.
Topics: Animals; Dexamethasone; Dose-Response Relationship, Drug; Hot Temperature; Inosine; Liver Neoplasms, Experimental; Methylthioinosine; Rats; Tyrosine Transaminase
PubMed: 6141163
DOI: No ID Found -
Inflammatory Bowel Diseases Jun 2010This study compared youth and parent-proxy reports of health-related quality of life (HRQoL) among youth with inflammatory bowel disease (IBD) to published comparison... (Comparative Study)
Comparative Study
BACKGROUND
This study compared youth and parent-proxy reports of health-related quality of life (HRQoL) among youth with inflammatory bowel disease (IBD) to published comparison group data and examined concordance between youth and parent-proxy reports of HRQoL.
METHODS
One hundred thirty-six youth and parent-proxy reports on the PedsQL 4.0 Generic Core Scales were compared to published data from chronically ill, acutely ill, and healthy comparison groups using independent samples t-tests. Reporter agreement was examined using paired samples t-tests and intraclass correlations (ICCs).
RESULTS
Youth with IBD reported lower psychosocial functioning than the healthy comparison group, higher physical and social functioning than the chronically ill group, and lower school functioning than all published comparison groups. Parent-proxy reports of youth HRQoL were higher than the chronically ill group, but lower than the healthy group on all scales except psychosocial functioning. Youth with active IBD reported lower physical health domain scores than youth with inactive disease. Concordance between youth and parent-proxy reports was moderate, with the lowest agreement in school and social functioning.
CONCLUSIONS
Youth with IBD and their parents rate HRQoL as lower than healthy youth but do not perceive the impact of IBD to be as limiting as in other chronic conditions. Youth report suggests that IBD may be particularly detrimental to HRQoL in the school functioning domain. Moderate agreement between parent and youth reports substantiates continued use of multiple informants in studies of pediatric HRQoL.
Topics: Adolescent; Adult; Child; Female; Humans; Inflammatory Bowel Diseases; Interpersonal Relations; Male; Mercaptopurine; Mesalamine; Methylthioinosine; Middle Aged; Parents; Quality of Life; Self Concept; Severity of Illness Index; Sickness Impact Profile; Social Adjustment; Thioguanine
PubMed: 19998462
DOI: 10.1002/ibd.21128 -
PloS One 2011Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have...
Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have previously shown that a thiopurine drug, 6-methylmercaptopurine riboside (6MMPr), inhibits replication of distantly related viruses within the Flaviviridae family in cell culture, including bovine viral diarrhea virus and hepatitis C virus replicon. Here we further examined the potential antiviral effect of 6MMPr on several diverse flaviviruses. In cell culture, 6MMPr inhibited virus production of yellow fever virus, dengue virus-2 (DENV-2) and West Nile virus (WNV) in a dose-dependent manner, and DENV-2 was significantly more sensitive to 6MMPr treatment than WNV. We then explored the use of 6MMPr as an antiviral against WNV in an immunocompetent mouse model. Once a day treatment of mice with 0.5 mg 6MMPr was just below the toxic dose in our mouse model, and this dose was used in subsequent studies. Mice were treated with 6MMPr immediately after subcutaneous inoculation with WNV for eight consecutive days. Treatment with 6MMPr exacerbated weight loss in WNV-inoculated mice and did not significantly affect mortality. We hypothesized that 6MMPr has low bioavailability in the central nervous system (CNS) and examined the effect of pre-treatment with 6MMPr on viral loads in the periphery and CNS. Pre-treatment with 6MMPr had no significant effect on viremia or viral titers in the periphery, but resulted in significantly higher viral loads in the brain, suggesting that the effect of 6MMPr is tissue-dependent. In conclusion, despite being a potent inhibitor of flaviviruses in cell culture, 6MMPr was not effective against West Nile disease in mice; however, further studies are warranted to reduce the toxicity and/or improve the bioavailability of this potential antiviral drug.
Topics: Animals; Antiviral Agents; Cells, Cultured; Chlorocebus aethiops; Cricetinae; Methylthioinosine; Mice; Vero Cells; Virus Replication; West Nile virus
PubMed: 22039536
DOI: 10.1371/journal.pone.0026697 -
The Journal of Biological Chemistry Aug 2005Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum...
Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.
Topics: Animals; Aspartic Acid; Cattle; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Plasmodium falciparum; Polyamines; Protein Binding; Protein Structure, Tertiary; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity; T-Lymphocytes
PubMed: 15961383
DOI: 10.1074/jbc.M505033200 -
The Journal of Biological Chemistry Apr 2004Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of... (Comparative Study)
Comparative Study
Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP.Immucillin-H (ImmH).SO(4) reveals a homohexamer with ImmH and SO(4) bound at each catalytic site. A solvent-filled cavity close to the 5'-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5'-carbon. Assays established 5'-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5'-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with K(d) values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP.MT-ImmH.SO(4) shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5'-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action.
Topics: Animals; Catalysis; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Methylthioinosine; Molecular Structure; Plasmodium falciparum; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles
PubMed: 14982926
DOI: 10.1074/jbc.C400068200 -
The Journal of Biological Chemistry Jun 1992The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and...
The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.
Topics: Amidophosphoribosyltransferase; Aminoimidazole Carboxamide; Animals; Azaserine; Folic Acid Antagonists; Leukemia, Experimental; Methotrexate; Methylthioinosine; Mice; Purines; Pyrimidines; Ribonucleotides; Tumor Cells, Cultured
PubMed: 1597445
DOI: No ID Found -
Biochemical Pharmacology May 19966-mercaptopurine (6MP) cytotoxicity is caused by thioguanine and methylthioinosine nucleotides. Thiopurine methylation occurs to a large extent in vivo and in vitro. In...
6-mercaptopurine (6MP) cytotoxicity is caused by thioguanine and methylthioinosine nucleotides. Thiopurine methylation occurs to a large extent in vivo and in vitro. In this reaction, S-adenosyl-L-methionine (AdoMet), produced from methionine and ATP, is converted into S-adenosyl-L-homocysteine (AdoHcy) which, in turn, is hydrolyzed into homocysteine. Remethylation of homocysteine into methionine is inhibited by methotrexate (MTX). In cultured lymphoblasts, AdoMet: AdoHcy ratio and DNA methylation decrease after incubation with 6MP. The aim of the present study was to investigate the influence of high-dose 6MP on the methylation capacity in children with acute lymphoblastic leukemia. Five patients received 4 courses with high-dose intravenous MTX (5' g.m-2 in 24 hr) immediately followed by high-dose 6MP (1300 mg.m-2 in 24 hr). Five control patients received high-dose MTX and oral 6MP (25 mg.m -2 daily for 8 weeks). Leucovorin rescue was started at 36 hr in both groups. In the intravenous 6MP group, 6-methylmercaptopurine, its riboside, and 6-methylmercapto-8-hydroxypurine were detectable in plasma in concentrations of 0.3-2.6 muM (6MP steady state levels: 11.6 muM). In red blood cells, mean methylthioinosine nucleotide levels were one third of those of ATP (13.1 nmol/10(8)). AdoHcy levels (10 pmol/10(8)) remained constant in both groups and AdoMet was not detectable ( < 20 pmol/10(8)). In both groups, plasma homocysteine increased and methionine decreased following administration of MTX. The delay in the recovery of methionine in the intravenous 6MP group after MTX infusion is probably the result of an increased demand on methyl groups during 6MP infusion.
Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Erythrocytes; Homocysteine; Humans; Mercaptopurine; Methionine; Methotrexate; Methylation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleotides; S-Adenosylhomocysteine; S-Adenosylmethionine
PubMed: 8645339
DOI: 10.1016/0006-2952(96)00032-9 -
British Journal of Clinical Pharmacology Jan 2008Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of...
AIMS
Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5'-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines.
METHODS
IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5'-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blood cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics.
RESULTS
A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg(-1) protein h(-1)) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (r(s) = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations.
CONCLUSION
Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism.
Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Biomarkers; Female; Humans; IMP Dehydrogenase; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Polymorphism, Genetic
PubMed: 17662091
DOI: 10.1111/j.1365-2125.2007.02985.x -
European Journal of Biochemistry Feb 19815'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and...
5'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and 5'-methylthioribose, has been purified to homogeneity from Lupinus luteus seeds. The nucleosidase has a native molecular weight of 62 000 and consists of two identical subunits, as judged by gel filtration and dodecylsulfate/polyacrylamide gel electrophoresis. The nucleosidase exhibits highest specificity towards the natural substrate with a Km of 4.1 X 10(-7) M for 5'-methylthioadenosine. It does not cleave adenine from S-adenosylhomocysteine. Among the synthetic analogs of 5'-methylthioadenosine tested, eleven compounds appear to be able to substitute as substrates. Furthermore, the enzyme can liberate hypoxanthinine from six inosyl (deaminated) derivatives obtained by enzymatic deamination of 5'-methylthioadenosine and its synthetic analogs. The Km for 5'-methylthioinosine is 55 microM, and the maximal velocity about 50-times lower than for 5'-methylthioadenosine. The reaction catalyzed by the nucleosidase can be inhibited by adenine (Ki = 11 microM), 3-deazaadenine (Ki = 19 microM), and 9-erythro(2-hydroxyl-3-nonyl)adenine (ki = 37 microM).
Topics: Adenosine; Kinetics; Molecular Weight; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Seeds; Substrate Specificity; Thionucleosides
PubMed: 6783408
DOI: 10.1111/j.1432-1033.1981.tb05148.x