Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

PubMed

Rieger, Hannah; Schmidt, Patrik; Schaeffeler, Elke; Abe, Manabu; Schiffhauer, Mira; Schwab, Matthias; von Ahsen, Nicolas; Zurek, Gabriela; Kirchherr, Hartmut; Shipkova, Maria; Wieland, Eberhard

2018-04-25

Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Thiopurines Induce Oxidative Stress in T-Lymphocytes: A Proteomic Approach

PubMed Central

Misdaq, Misbah; Ziegler, Sonia; von Ahsen, Nicolas; Asif, Abdul R.

2015-01-01

Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients. PMID:25873760

Pharmacogenetics during standardised initiation of thiopurine treatment in inflammatory bowel disease

PubMed Central

Hindorf, U; Lindqvist, M; Peterson, C; Söderkvist, P; Ström, M; Hjortswang, H; Pousette, A; Almer, S

2006-01-01

Background Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. Aim To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. Patients 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. Methods Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6‐mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. Results 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)); 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio; p = 0.02). Patients with meTIMP concentrations >11 450 pmol/8×108 red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0; p = 0.015). Conclusions After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity. PMID:16543290

A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia

PubMed Central

Yang, Suk-Kyun; Hong, Myunghee; Baek, Jiwon; Choi, Hyunchul; Zhao, Wanting; Jung, Yusun; Haritunians, Talin; Ye, Byong Duk; Kim, Kyung-Jo; Park, Sang Hyoung; Park, Soo-Kyung; Yang, Dong-Hoon; Dubinsky, Marla; Lee, Inchul; McGovern, Dermot P B; Liu, Jianjun; Song, Kyuyoung

2016-01-01

Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn’s disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p.Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; Pcombined = 4.88 × 10−94). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10−4). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations. PMID:25108385

A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations.

PubMed

Coulthard, S A; Rabello, C; Robson, J; Howell, C; Minto, L; Middleton, P G; Gandhi, M K; Jackson, G; McLelland, J; O'Brien, H; Smith, S; Reid, M M; Pearson, A D; Hall, A G

2000-09-01

S-Methylation by thiopurine methyltransferase (TPMT) is an important route of metabolism for the thiopurine drugs. About one in 300 individuals are homozygous for a TPMT mutation associated with very low enzyme activity and severe myelosuppression if treated with standard doses of drug. To validate the use of molecular genetic techniques for the detection of TPMT deficiency, we have determined red blood cell TPMT activity in 240 adult blood donors and 55 normal children. Genotype was determined by restriction fragment length analysis of polymerase chain reaction products in a cohort of 79 of the blood donors and five cases of azathioprine-induced myelosupression, and this confirmed a close relationship between genotype and phenotype. In 17 of the 24 cases in which mutations were found, DNA was also available from remission bone marrow. In one of these cases, DNA from the remission marrow sample indicated the presence of a non-mutated allele that had not been seen in the blast DNA sample obtained at presentation. These results indicate that polymerase chain reaction-based assays give reliable and robust results for the detection of TPMT deficiency, but that caution should be exercised in relying exclusively on DNA obtained from lymphoblasts in childhood leukaemia.

Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid chromatography hyphenated with mass spectrometry.

PubMed

Pecher, Daniel; Dokupilová, Svetlana; Zelinková, Zuzana; Peppelenbosch, Maikel; Mikušová, Veronika; Mikuš, Peter

2017-08-05

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry (MS). In comparison with reversed-phase high-performance liquid chromatography (RP-HPLC), that has been typically applied in determination of TPMT activity, the HILIC significantly improved the analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution. The HILIC-HPLC-MS method was optimized and validated, providing favorable parameters of detection and quantitation limits (5.5 and 16.5pmol/mL, respectively), linearity (coefficient of determination 0.9999 in the range of 0.01-1.0nmol/mL), recovery and precision (93.25-100.37% with RSD 1.06-1.32% in the whole concentration range of QC samples). Moreover, in contrast to the conventional RP-HPLC-UV approach, the complex phenotype TPMT profiles can be reliably and without interferences monitored using the HILIC-HPLC-MS method. Such advanced monitoring can provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non-methylated 6-mercaptopurine) and, by that, TPMT action in biological systems before and during the therapy of IBD. Copyright © 2017 Elsevier B.V. All rights reserved.

Methylation of 6-mercaptopurine and 6-thioguanine by thiopurine S-methyltransferase. A comparison of activity in red blood cell samples of 199 blood donors.

PubMed

Kröplin, T; Iven, H

2000-07-01

To compare 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrates for the methylation reaction catalysed by the enzyme thiopurine S-methyltransferase (TPMT). TPMT activity in haemolysed red blood cells of healthy blood donors was determined twice, using the same experimental setting and equal molar concentrations of 6-TG and 6-MP as substrates. After extraction, the reaction products 6-methyl-TG and 6-methyl-MP were quantified using specific high-performance liquid chromatography procedures. The medians of the TPMT activities from 199 blood donors were 54.4 nmol 6-MTG g(-1)Hb h(-1) when measured with 6-TG as the substrate and 35.8 nmol 6-MMP g(-1) Hb h(-1) when measured with 6-MP. The correlation coefficient for the 199 pairs of values was 0.8695. On average, TPMT activity was 34% lower with 6-MP as substrate than with 6-TG as substrate.

Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

PubMed Central

Jacqz-Aigrain, E; Bessa, E; Medard, Y; Mircheva, Y; Vilmer, E

1994-01-01

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively). PMID:7946931

Thiopurine methyltransferase and treatment outcome in the UK acute lymphoblastic leukaemia trial ALL2003.

PubMed

Lennard, Lynne; Cartwright, Cher S; Wade, Rachel; Vora, Ajay

2015-08-01

The influence of thiopurine methyltransferase (TPMT) genotype on treatment outcome was investigated in the United Kingdom childhood acute lymphoblastic leukaemia trial ALL2003, a trial in which treatment intensity was adjusted based on minimal residual disease (MRD). TPMT genotype was measured in 2387 patients (76% of trial entrants): 2190 were homozygous wild-type, 189 were heterozygous for low activity TPMT alleles (166 TPMT*1/*3A, 19 TPMT*1/*3C, 3 TPMT*1/*2 and 1 TPMT*1/*9) and 8 were TPMT deficient. In contrast to the preceding trial ALL97, there was no difference in event-free survival (EFS) between the TPMT genotypes. The 5-year EFS for heterozygous TPMT*1/*3A patients was the same in both trials (88%), but for the homozygous wild-type TPMT*1/*1 patients, EFS improved from 80% in ALL97% to 88% in ALL2003. Importantly, the unexplained worse outcome for heterozygous TPMT*1/*3C patients observed in ALL97 (5-year EFS 53%) was not seen in ALL2003 (5-year EFS 94%). In a multivariate Cox regression analysis the only significant factor affecting EFS was MRD status (hazard ratio for high-risk MRD patients 4·22, 95% confidence interval 2·97-5·99, P < 0·0001). In conclusion, refinements in risk stratification and treatment have reduced the influence of TPMT genotype on treatment outcome in a contemporary protocol. © 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.

Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells.

PubMed

Karim, Hazhar; Ghalali, Aram; Lafolie, Pierre; Vitols, Sigurd; Fotoohi, Alan K

2013-07-26

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug. Copyright © 2013 Elsevier Inc. All rights reserved.

The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase.

PubMed

Hall, A G; Hamilton, P; Minto, L; Coulthard, S A

2001-01-30

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.

Thiopurine methyltransferase polymorphisms and mercaptopurine tolerance in Turkish children with acute lymphoblastic leukemia.

PubMed

Albayrak, Meryem; Konyssova, Uljan; Kaya, Zuhre; Gursel, Turkiz; Guntekin, Sezen; Percin, E Ferda; Kocak, Ulker

2011-11-01

Thiopurine methyltransferase (TPMT) enzyme is involved in the metabolism of 6-mercaptopurine (6-MP), a key component of acute lymphoblastic leukemia (ALL) treatment protocols in children. The aims of this study were to investigate the frequency of common genetic polymorphisms associated with low TPMT activity and correlations of polymorphic variants with 6-MP tolerance in a group of Turkish children with ALL. Genotyping for G238C, A719G, and G460A mutations were performed by using NanoChip Technology. Adverse reactions during the first 6 months of maintenance therapy with oral 6-MP and methotrexate were retrospectively analyzed from patient's files. Five (8.6%) of 58 children with ALL had a polymorphic TPMT allele: 4 (3.4%) were heterozygous for TPMT*3A (G460A and A719G), and one (0.9%) was heterozygous for TPMT*3C (A719G). No cases with TPMT*3B (G460A) or TPMT*2 (G238C) variants were identified. Children with TPMT*3A and *3C had significantly lower leukocyte and neutrophil counts and percentage of target 6-MP dosage, and longer periods with ≥grade 2 infections, ≥grade 2 liver toxicity, and chemotherapy interruptions than the children with wild-type TPMT during the first 24 weeks of maintenance therapy. The frequency and distribution of common TPMT polymorphisms in Turkish children with ALL is similar to other Caucasian populations. Polymorphic variants were associated with excessive 6-MP toxicity supporting the suggestion that TPMT genotyping should be performed before institution of 6-MP therapy.

Whole-Blood Thiopurine S-Methyltransferase Genotype and Phenotype Concordance in Iranian Kurdish Ulcerative Colitis (UC) Patients.

PubMed

Bahrehmand, Fariborz; Vaisi-Raygani, Asad; Kiani, Amir; Bashiri, Homayoun; Zobeiri, Mahdi; Tanhapour, Maryam; Pourmotabbed, Tayebeh

2017-05-01

Thiopurine methyl transferase (TPMT), a drug-metabolizing enzyme, catalyzes methylation and consequently, the metabolism of thiopurine compounds used for treatment of inflammatory bowel disease (IBD). Individuals who are homozygous recessive or have extremely low TPMT activity need to avoid thiopurines because of concern for significant leukopenia. The aim of this research was to determine TPMT phenotypes and genotypes in IBD patients to predict the risk of thiopurine toxicity before treatment. The present case-control study consisted of 210 ulcerative colitis patients and 212 unrelated healthy controls from the population of western Iran. TPMT phenotype and genotype were determined by HPLC and allele specific PCR and PCR-RFLP, respectively. TPMT phenotyping and genotyping were compatible and demonstrated no frequency for deficient, 2.2% for low, and 97.8% for normal-activity which is different compared with the results of other studies. There was a significant negative correlation between TPMT activities as calculated based on nmol6MTG/gHb/h and the Hb levels in both UC (r = -0.54, p < 0.001) and control groups (r = -0.27, p < 0.001). Interestingly, a significant positive correlation between Hb levels and TPMT activities was seen when the enzyme activity was calculated in mU/L in both UC patients (r = 0.14, p = 0.05) and in control subjects (r = 0.43, p < 0.001). The overall concordance rate between TPMT phenotypes and genotypes of mutants to alleles (9 out of 422), based on receiver-operating characteristic (ROC) curve, yielded a sensitivity of 94.7% and specificity of 90% for mU/L and a sensitivity of 85.6% and specificity of 90% for nmol6MTG/gHb/h. The use of mU/L is more appropriate than nmol6MTG/gHb/h for expressing TPMT activity, and there is better correlation between genotypes and phenotypes of TPMT based on mU/L. The frequency of known mutant TPMT alleles in western Iran (Kurd population) is low suggesting low risk of thiopurine drug toxicity in IBD patients from this region.

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

PubMed

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Analysis of Thiopurine S-Methyltransferase Deficient Alleles in Acute Lymphoblastic Leukemia Patients in Mexican Patients.

PubMed

Jiménez-Morales, Silvia; Ramírez-Florencio, Mireya; Mejía-Aranguré, Juan Manuel; Núñez-Enríquez, Juan Carlos; Bekker-Mendez, Carolina; Torres-Escalante, José Luis; Flores-Lujano, Janet; Jiménez-Hernández, Elva; Del Carmen Rodríguez-Zepeda, María; Leal, Yelda A; González-Montalvo, Pablo Miguel; Pantoja-Guillen, Francisco; Peñaloza-Gonzalez, José Gabriel; Gutiérrez-Juárez, Erick Israel; Núñez-Villegas, Nora Nancy; Pérez-Saldivar, Maria Luisa; Guerra-Castillo, Francisco Xavier; Flores-Villegas, Luz Victoria; Ramos-Cervantes, María Teresa; Fragoso, José Manuel; García-Escalante, María Guadalupe; Del Carmen Pinto-Escalante, Doris; Ramírez-Bello, Julián; Hidalgo-Miranda, Alfredo

2016-11-01

It has been demonstrated that heterozygote and homozygote thiopurine S-methyltransferase (TPMT) mutant allele carriers are at high risk to develop severe and potentially fatal hematopoietic toxicity after treatment with standard doses of 6-mercaptopurine (6-MP) and methotrexate (MX). Those drugs are the backbone of acute lymphoblastic leukemia (ALL) and several autoimmune disease treatments. We undertook this study to determine the frequency of the TPMT deficient alleles in children with ALL and non-ALL subjects from Mexico City and Yucatan, Mexico. We included 849 unrelated subjects, of which 368 ALL children and 342 non-ALL subjects were from Mexico City, and 60 ALL cases and 79 non-ALL individuals were from Yucatan. Genotyping of the rs1800462, rs1800460 and rs1142345 SNPs was performed by 5'exonuclease technique using TaqMan probes (Life Technologies Foster City, CA). The mutant TPMT alleles were present in 4.8% (81/1698 chromosomes) and only 0.2% were homozygote TPMT*3A/TPMT*3A. We did not find statistically significant differences in the distribution of the mutant alleles between patients from Mexico City and Yucatan in either ALL cases or non-ALL. Nonetheless, the TPMT*3C frequency in ALL patients was higher than non-ALL subjects (p = 0.03). To note, the null homozygous TPMT*3A/TPMT*3A genotype was found in 2.5% of the non-ALL subjects. TPMT mutant alleles did not exhibit differential distribution between both evaluated populations; however, TPMT*3C is overrepresented in ALL cases in comparison with non-ALL group. Assessing the TPMT mutant alleles could benefit the ALL children and those undergoing 6-MP and MX treatment. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

Should we test TPMT enzyme levels before starting azathioprine?

PubMed

Richard, Vijay Samuel; Al-Ismail, Deana; Salamat, Ahmed

2007-08-01

Thiopurine methyltransferase (TPMT) is the main enzyme responsible for inactivating toxic products of azathioprine (AZA) metabolism. Patients with homozygous deficiency of this enzyme have no enzyme activity and ideally should not be given AZA. Patients with heterozygous deficiency have 50% of enzyme activity and have been shown to respond well and tolerate half a standard dose. We describe a patient with homozygous deficiency of TPMT who developed life threatening neutropenic sepsis, and advocate that all patients should be tested for TPMT activity prior to starting AZA therapy.

Assessment of thiopurine methyltransferase activity in patients prescribed azathioprine or other thiopurine-based drugs.

PubMed

Booth, Ronald A; Ansari, Mohammed T; Tricco, Andrea C; Loit, Evelin; Weeks, Laura; Doucette, Steve; Skidmore, Becky; Hoch, Jeffrey S; Tsouros, Sophia; Sears, Margaret; Sy, Richmond; Karsh, Jacob; Mani, Suja; Galipeau, James; Yurkiewich, Alexander; Daniel, Raymond; Tsertsvadze, Alexander; Yazdi, Fatemeh

2010-12-01

To examine whether pretreatment determination of thiopurine methyltransferase (TPMT) enzymatic activity (phenotyping) or TPMT genotype, to guide thiopurine therapy in chronic autoimmune disease patients, reduces treatment harms. Other objectives included assessing: preanalytic, analytic, and postanalytic requirements for TPMT testing; diagnostic accuracy of TPMT genotyping versus phenotyping; association of thiopurine toxicity with TPMT genotypic or phenotypic status; and costs of testing, care, and treating drug-associated complications. MEDLINE®, EMBASE®, and Healthstar were searched from inception to May 2010; the Cochrane Library® to October 2009; and BIOSIS®, Genetics Abstracts, and EconLit™ to May 2009, for English language records. A reviewer screened records, and a second reviewer verified exclusions and subsequent selection of relevant studies. Studies in patients with leukemia and organ transplant were excluded. Additionally, laboratories that provide TPMT analytical services were surveyed to assess means of TPMT testing in practice. Where possible, risk of bias was assessed using standard criteria. Meta-analyses estimated diagnostic sensitivity, and specificity; and odds ratios of associations. 1790 titles or abstracts, and 538 full text records were screened. 114 observational studies and one RCT were included. Majority of studies were rated fair quality, except for diagnostic studies with 37 percent of studies rated poor. In general, there were few patients who were homozygous (or compound heterozygous) for TPMT variant alleles in the included studies limiting applicability. There is insufficient evidence examining effectiveness of pretesting in terms of reduction in clinical adverse events. Sufficient preanalytical data were available regarding preferred specimen collection, stability and storage conditions for TPMT testing. There was no clinically significant effect of age, gender, various coadministered drugs, or most morbidities (with the exception of renal failure and dialysis). TPMT phenotyping methods had coefficients of variation generally below 10 percent. TPMT genotyping reproducibility is generally between 95-100 percent. The sensitivity of genotyping to identify patients with low or intermediate TPMT enzymatic activity is imprecise, ranging from 70.70 to 82.10 percent (95 percent CI, lower bound range 37.90 to 54.00 percent; upper bound range 84.60 to 96.90 percent). Sensitivity of homozygous TPMT genotype to correctly identify patients with low to absent enzymatic activity was 87.10 percent (95 percent CI 44.30 to 98.30 percent). Genotyping specificity approached 100 percent. Leukopenia was significantly associated with low and intermediate enzymatic activity (low activity OR 80.00, 95 percent CI 11.5 to 559; and intermediate activity OR 2.96, 95 percent CI 1.18 to 7.42), and homozygous and heterozygous TPMT variant allele genotype (OR 18.60, 95 percent CI 4.12 to 83.60; and 4.62, 95 percent CI 2.34 to 9.16, respectively). In general, TPMT phenotyping costs less than genotyping, although estimates across studies are quite heterogeneous. There is insufficient direct evidence regarding the effectiveness of pretesting of TPMT status in patients with chronic autoimmune diseases. Indirect evidence confirms strong association of leukopenia with lower levels of TPMT activity and carrier genotype already established in the literature.

Assessment of thiopurine methyltransferase activity in patients prescribed azathioprine or other thiopurine-based drugs.

PubMed Central

Booth, Ronald A; Ansari, Mohammed T; Tricco, Andrea C; Loit, Evelin; Weeks, Laura; Doucette, Steve; Skidmore, Becky; Hoch, Jeffrey S; Tsouros, Sophia; Sears, Margaret; Sy, Richmond; Karsh, Jacob; Mani, Suja; Galipeau, James; Yurkiewich, Alexander; Daniel, Raymond; Tsertsvadze, Alexander; Yazdi, Fatemeh

2010-01-01

OBJECTIVES To examine whether pretreatment determination of thiopurine methyltransferase (TPMT) enzymatic activity (phenotyping) or TPMT genotype, to guide thiopurine therapy in chronic autoimmune disease patients, reduces treatment harms. Other objectives included assessing: preanalytic, analytic, and postanalytic requirements for TPMT testing; diagnostic accuracy of TPMT genotyping versus phenotyping; association of thiopurine toxicity with TPMT genotypic or phenotypic status; and costs of testing, care, and treating drug-associated complications. DATA SOURCES MEDLINE®, EMBASE®, and Healthstar were searched from inception to May 2010; the Cochrane Library® to October 2009; and BIOSIS®, Genetics Abstracts, and EconLit™ to May 2009, for English language records. REVIEW METHODS A reviewer screened records, and a second reviewer verified exclusions and subsequent selection of relevant studies. Studies in patients with leukemia and organ transplant were excluded. Additionally, laboratories that provide TPMT analytical services were surveyed to assess means of TPMT testing in practice. Where possible, risk of bias was assessed using standard criteria. Meta-analyses estimated diagnostic sensitivity, and specificity; and odds ratios of associations. RESULTS 1790 titles or abstracts, and 538 full text records were screened. 114 observational studies and one RCT were included. Majority of studies were rated fair quality, except for diagnostic studies with 37 percent of studies rated poor. In general, there were few patients who were homozygous (or compound heterozygous) for TPMT variant alleles in the included studies limiting applicability. There is insufficient evidence examining effectiveness of pretesting in terms of reduction in clinical adverse events. Sufficient preanalytical data were available regarding preferred specimen collection, stability and storage conditions for TPMT testing. There was no clinically significant effect of age, gender, various coadministered drugs, or most morbidities (with the exception of renal failure and dialysis). TPMT phenotyping methods had coefficients of variation generally below 10 percent. TPMT genotyping reproducibility is generally between 95-100 percent. The sensitivity of genotyping to identify patients with low or intermediate TPMT enzymatic activity is imprecise, ranging from 70.70 to 82.10 percent (95 percent CI, lower bound range 37.90 to 54.00 percent; upper bound range 84.60 to 96.90 percent). Sensitivity of homozygous TPMT genotype to correctly identify patients with low to absent enzymatic activity was 87.10 percent (95 percent CI 44.30 to 98.30 percent). Genotyping specificity approached 100 percent. Leukopenia was significantly associated with low and intermediate enzymatic activity (low activity OR 80.00, 95 percent CI 11.5 to 559; and intermediate activity OR 2.96, 95 percent CI 1.18 to 7.42), and homozygous and heterozygous TPMT variant allele genotype (OR 18.60, 95 percent CI 4.12 to 83.60; and 4.62, 95 percent CI 2.34 to 9.16, respectively). In general, TPMT phenotyping costs less than genotyping, although estimates across studies are quite heterogeneous. CONCLUSIONS There is insufficient direct evidence regarding the effectiveness of pretesting of TPMT status in patients with chronic autoimmune diseases. Indirect evidence confirms strong association of leukopenia with lower levels of TPMT activity and carrier genotype already established in the literature. PMID:23126559

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

PubMed

Tai, H L; Krynetski, E Y; Yates, C R; Loennechen, T; Fessing, M Y; Krynetskaia, N F; Evans, W E

1996-04-01

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.

Congenital thiopurine methyltransferase deficiency and 6-mercaptopurine toxicity during treatment for acute lymphoblastic leukaemia.

PubMed Central

Lennard, L; Gibson, B E; Nicole, T; Lilleyman, J S

1993-01-01

Two children with acute lymphoblastic leukaemia (ALL) taking daily 6-mercaptopurine as part of a national UK therapeutic trial repeatedly developed profound myelosuppression on 25% of the standard protocol dose. Both were found to have undetectable intracellular activity of thiopurine methyltransferase (TPMT), an enzyme controlling one of the major alternative catabolic pathways of 6-mercaptopurine, and both produced higher concentrations of cytotoxic drug metabolites at 10-25% of the protocol dose than other patients taking 100%. It is supposed that these patients represent the 0.33% of the normal population constitutionally lacking TPMT. It is important to recognise such individuals both to avoid fatal bone marrow failure through inadvertent overdosage, and to be reassured that an adequate drug effect can be achieved at around 10% of the standard dose. PMID:8257179

Myelotoxicity after high-dose methotrexate in childhood acute leukemia is influenced by 6-mercaptopurine dosing but not by intermediate thiopurine methyltransferase activity.

PubMed

Levinsen, Mette; Rosthøj, Susanne; Nygaard, Ulrikka; Heldrup, Jesper; Harila-Saari, Arja; Jonsson, Olafur G; Bechensteen, Anne Grete; Abrahamsson, Jonas; Lausen, Birgitte; Frandsen, Thomas L; Weinshilboum, Richard M; Schmiegelow, Kjeld

2015-01-01

Through enhancement of 6-mercaptopurine (6MP) bioavailability and inhibition of purine de novo synthesis, high-dose methotrexate (HD-MTX) may increase incorporation into DNA of 6-thioguanine nucleotides, the cytotoxic metabolites of 6MP. Patients with intermediate activity of thiopurine methyltransferase (TPMT(IA)) have higher cytosol 6-thioguanine nucleotide levels. We investigated toxicity following HD-MTX during MTX/6MP maintenance therapy in relation to 6MP and TPMT. Using linear mixed models, we explored myelo- and hepatotoxicity in relation to 6MP dosage and TPMT phenotype following 1,749 HD-MTX courses to 411 children with acute lymphoblastic leukemia on maintenance therapy. The degree of myelosuppression following HD-MTX was similar for patients with TPMT(IA) and patients with high TPMT activity (TPMT(HA)), when HD-MTX started with same blood counts and 6MP doses. However, since TPMT(IA) had lower blood counts at initiation of HD-MTX compared with TPMT(HA) patients (median WBC 2.8 vs. 3.3 × 10⁹/L, P = 0.01; median ANC 1.4 vs. 1.7 × 10⁹/L, P = 0.02), TPMT(IA) continued to have lower WBC and ANC levels compared with TPMT(HA) during all 28 days after HD-MTX [relative difference 9 % (95 % CI 2-17), P = 0.02 and 21 % (95 % CI 6-39), P = 0.005]. Still, the fractional decrease in WBC and ANC levels after HD-MTX did not differ between TPMT(IA) and TPMT(HA) patients (P = 0.47; P = 0.38). The degree of leukopenia, neutropenia, thrombocytopenia and rise in aminotransferases were all significantly related to 6MP dose (P < 0.001 for all analyses). For both TPMT(IA) and TPMT(HA) patients, dose of 6MP prior to HD-MTX should be guided by pre-HD-MTX blood counts, but not by TPMT activity.

Bioequivalence of azathioprine products.

PubMed

Baker, Daniel E

2003-01-01

All azathioprine oral tablets are considered bioequivalent by the Food and Drug Administration based on traditional testing. However, since these tests were conducted, it has been determined that some patients have a deficiency of the enzyme most responsible for the metabolism of 6-mercaptopurine-thiopurine methyltransferase (TPMT). Azathioprine is rapidly converted to 6-mercaptopurine, its active metabolite. So it is possible that differences in TPMT activity may influence the bioequivalence of azathioprine products among individuals, especially those patients deficient in TPMT enzyme activity. However, this possibility has not been evaluated.

Differential effects of thiopurine methyltransferase (TPMT) and multidrug resistance-associated protein gene 4 (MRP4) on mercaptopurine toxicity.

PubMed

Liu, Chengcheng; Janke, Laura J; Yang, Jun J; Evans, William E; Schuetz, John D; Relling, Mary V

2017-08-01

Mercaptopurine plays a pivotal role in treatment of acute lymphoblastic leukemia (ALL) and autoimmune diseases, and inter-individual variability in mercaptopurine tolerance can influence treatment outcome. Thiopurine methyltransferase (TPMT) and multi-drug resistant Protein 4 (MRP4) have both been associated with mercaptopurine toxicity in clinical studies, but their relative contributions remain unclear. We studied the metabolism of and tolerance to mercaptopurine in murine knockout models of Tpmt, Mrp4, and both genes simultaneously. Upon mercaptopurine treatment, Tpmt -/- Mrp4 -/- mice had the highest concentration of bone marrow thioguanine nucleotides (8.5 pmol/5 × 10 6 cells, P = 7.8 × 10 -4 compared with 2.7 pmol/5 × 10 6 cells in wild-types), followed by those with Mrp4 or Tpmt deficiency alone (6.1 and 4.3 pmol/5 × 10 6 cells, respectively). Mrp4-deficient mice accumulated higher concentrations of methylmercaptopurine metabolites compared with wild-type (76.5 vs. 23.2 pmol/5 × 10 6 cells, P = 0.027). Mice exposed to a clinically relevant mercaptopurine dosing regimen displayed differences in toxicity and survival among the genotypes. The double knock-out of both genes experienced greater toxicity and shorter survival compared to the single knockout of either Tpmt (P = 1.7 × 10 -6 ) or Mrp4 (P = 7.4 × 10 -10 ). We showed that both Tpmt and Mrp4 influence mercaptopurine disposition and toxicity.

Epistatic interactions between thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) variations determine 6-mercaptopurine toxicity in Indian children with acute lymphoblastic leukemia.

PubMed

Dorababu, Patchva; Nagesh, Narayana; Linga, Vijay Gandhi; Gundeti, Sadashivudu; Kutala, Vijay Kumar; Reddanna, Pallu; Digumarti, Raghunadharao

2012-04-01

To explore the role of genetic variants of thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) in 6-mercaptopurine (6-MP)-induced toxicity in Indian children with acute lymphoblastic leukemia (ALL). Children with ALL receiving 6-MP in maintenance phase of treatment (n = 90) were enrolled in the study. Bidirectional sequencing of TPMT (whole gene) and ITPA (exon 2, exon 3, and intron 2) was undertaken, and correlation between genotype and 6-MP toxicity was assessed. Five variations were observed in TPMT, including two exonic variations, TPMT*12 (374 C > T) and TPMT*3C (719A > G), and three intronic, intron 3 (12356 C > T), intron 4 (16638 C > T), and TPMT rs2842949. Two exonic, ITPA exon -2 (94 C → A) and exon 3 of ITPA (138 G > A), and one intronic, ITPA intron 2 (A→C), variations were observed in ITPA. Multifactor dimensionality reduction analysis of all the genetic variants showed independent association of ITPA 94 C→A as well as synergic epistatic interactions, i.e., TPMT*12 × ITPA ex3, ITPA ex2 × TPMT*12 × ITPA ex3, and TPMT*3C × ITPA ex2 × TPMT*12 × ITPA ex3, in determining hematological toxicity. This is further substantiated by a multiple linear regression model, which showed moderate predictability of toxicity with these variants (area under the curve = 0.70, p = 0.004). Our results suggest that apart from the individual effect of ITPA 94 C→A, epistatic interactions between the variations of TPMT (*3C, *12) and ITPA (ex2, ex3) are associated with the 6-MP toxicity. Testing these variants facilitates tailoring of the 6-MP therapy in children with ALL.

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

PubMed Central

Tai, H. L.; Krynetski, E. Y.; Yates, C. R.; Loennechen, T.; Fessing, M. Y.; Krynetskaia, N. F.; Evans, W. E.

1996-01-01

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8644731

TPMT Genetic Variations in Populations of the Russian Federation

PubMed Central

Samochatova, Elena V; Chupova, Natalia V; Rudneva, Anastassia; Makarova, Olga; Nasedkina, Tatyana V; Fedorova, Olga E; Glotov, Andrei S; Kozhekbaeva, Zh.; Maiorova, Olga A; Roumyantsev, Alexander G; Krynetski, Eugene Y; Krynetskaia, Natalia F; Evans, William E; Ribeiro, Raul C

2009-01-01

Background Polymorphisms that reduce the activity of thiopurine S-methyltransferase (TPMT) cause adverse reactions to conventional doses of thiopurines, routinely used for antileukemic and immunosuppressive treatment. There are more than 20 variant alleles of TPMT that cause decreased enzymatic activity. We studied the most common variant alleles of TPMT and their frequency distribution in a large cohort of multiracial residents in the Russian Federation and compared their frequencies in children with and without malignancy to determine whether TPMT gene abnormality is associated with hematologic malignancy. Procedure The TPMT biochip was used to detect 6 TPMT single nucleotide polymorphisms (SNPs) corresponding to 7 TPMT-deficiency alleles (TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8). We analyzed allele frequencies in the whole cohort, the childhood cancer group, and the non-cancer group. We also characterized disease features and outcome according to the presence of TPMT SNPs in children with acute lymphoblastic leukemia (ALL). Results Fifty-five (5.5%) study participants overall had heterozygous TPMT genotypes (1 variant and 1 wild-type allele): TPMT*1/*3A (n=45; 4.5%), TPMT*1/*3C (n=8; 0.8%), and TPMT*1/*2 (n=2; 0.2%). TPMT SNPs were more frequent in children with hematologic malignancy than in other participants (7.5% vs. 4.0%, P = 0.02). We found no significant association between TPMT SNPs and ALL treatment outcome (median follow-up, 31.3 months). Conclusions TPMT*3A is the most prevalent variant allele in the Russian Federation. The estimated frequency of variant alleles in the study cohort (5.5%) was similar to that observed in the White populations in the U.S. and Eastern Europe. PMID:19034904

Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements.

PubMed

Kröplin, T; Fischer, C; Iven, H

1999-06-01

Thiopurine S-methyltransferase (TPMT) activity, when measured in red blood cells (RBC) with a recently published TPMT activity assay using 6-thioguanine (6-TG) as substrate, could not be reproduced in another laboratory. We investigated factors which could influence the results of the TPMT activity measurement. We tested twelve 6-TG and four 6-mercaptopurine (6-MP) compounds from different suppliers as substrates and determined the enzyme kinetic parameters Km and Vmax. Furthermore, we studied the influence of different 6-TG compounds on the affinity of the methyl donor S-adenosyl-L-methionine (SAM) to the TPMT enzyme. All 6-TG products were of equal purity (declared >98% by the supplier): this was ascertained by HPLC. However, the rate of methylation obtained following incubation with 6-TG from different suppliers ranged from 10% to 100% when incubated with the same RBC lysate. The lowest apparent Km value for a 6-TG was 22.3 micromol x l(-1), while the product with the highest methylation rate showed a Km of 156 micromol x l(-1). From these results we assume that there is a contaminant in some 6-TG products, which acts as a strong inhibitor of TPMT activity. Compounds possibly used for the synthesis of 6-TG (guanine, pyridine, 6-chloroguanine) did not affect the methylation rate. Thioxanthine, which is known to be a strong inhibitor of TPMT when added to the assay system to give a 2% contamination, reduced TPMT activity from 100% to 72%. Using 6-MP from different suppliers as substrate resulted in Km values ranging from 110 to 162 micromol x l(-1) and Vmox values ranging from 54 to 68 nmol 6-MMP x g(-1)Hb x h(-1). The Km value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9-11 micromol-l(-1) SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 micromol x l(-1) SAM), but could become relevant in experiments using higher SAM concentrations. TPMT enzyme activity determined with 6-TG as substrate may be strongly inhibited by a contaminant in some of the 6-TG lots distributed.

The pharmacokinetic effect of discontinuation of mesalazine on mercaptopurine metabolite levels in inflammatory bowel disease patients.

PubMed

Gilissen, L P L; Bierau, J; Derijks, L J J; Bos, L P; Hooymans, P M; van Gennip, A; Stockbrügger, R W; Engels, L G J B

2005-10-01

In vitro studies suggest interactions between mesalazine (mesalamine) and thiopurines by thiopurine S-methyltransferase (TPMT) inhibition, influencing the balance of hepatotoxic 6-methylmercaptopurine ribonucleotide and immunosuppressive tioguanine (thioguanine) metabolites. To examine the in vivo pharmacokinetic interaction between mesalazine and mercaptopurine. A prospective study was performed in quiescent inflammatory bowel disease patients using the combination of mercaptopurine and mesalazine. Laboratory parameters, 6-methylmercaptopurine ribonucleotide and tioguanine levels and thiopurine S-methyltransferase activity in erythrocytes were measured at stable medication, after mesalazine discontinuation and mesalazine reintroduction, further mercaptopurine was continued. Seventeen patients were participated. Mean mercaptopurine dose was 0.78 mg/kg/day and median of mesalazine dose was 3000 mg/day. After mesalazine discontinuation, mean tioguanine levels changed significantly from 262 to 209 pmol/8 x 10(8) red blood cell, increasing to 270 after reintroduction. Mean 6-methylmercaptopurine ribonucleotide levels were 1422, 2149 and 1503 pmol/8 x 10(8) red blood cell respectively. Mean 6-methylmercaptopurine ribonucleotide/tioguanine ratio increased significantly from 6.3 at baseline to 11.2. Mean baseline thiopurine S-methyltransferase activity was 0.58 pmol/10(6) red blood cell/h and stable. All patients had wild-type thiopurine S-methyltransferase genotypes however, leucocyte counts were stable. A significantly higher tioguanine levels and improving 6-methylmercaptopurine ribonucleotide/tioguanine ratio were found during mesalazine/mercaptopurine combination. Theoretically, mesalazine inhibits thiopurine S-methyltransferase activity. In vivo thiopurine S-methyltransferase activity did not change, however. Mesalazine has synergistic effects on mercaptopurine therapy, but the mechanism is unclear. Combining these drugs may be further indication for mesalazine in inflammatory bowel disease treatment.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine

PubMed Central

Hawwa, Ahmed F; Millership, Jeff S; Collier, Paul S; Vandenbroeck, Koen; McCarthy, Anthony; Dempsey, Sid; Cairns, Carole; Collins, John; Rodgers, Colin; McElnay, James C

2008-01-01

AIMS To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). METHODS Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94→A and IVS2+21A→C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. RESULTS Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A→C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferaseheterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A→C variants with thrombocytopenia (P = 0.012). CONCLUSIONS Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT6-Mercaptopurine (6-MP) and azathioprine (AZA) are both inactive prodrugs that require intracellular activation into the active 6-thioguanine nucleotides (6-TGNs).This metabolic process undergoes three different competitive pathways that are catalysed by three different enzymes; xanthine oxidase (XO), thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA), all of which exhibit genetic polymorphisms.Although the impact of genetic variation in the TPMT gene on treatment outcome and toxicity has been demonstrated, the role of other polymorphisms remains less well known. WHAT THIS STUDY ADDS New information on the allelic variation of these three enzymes (XO, TPMT and ITPA) and their influence on 6-MP/AZA metabolism and toxicity.Confirmation of the association of TPMT polymorphism with haematological toxicity.Identified potential genetic characteristics that may contribute to higher risk of adverse events (such as ITPA IVS2+21A→C mutation). PMID:18662289

TPMT and MTHFR genotype is not associated with altered risk of thioguanine-related sinusoidal obstruction syndrome in pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group.

PubMed

Wray, Lisa; Vujkovic, Marijana; McWilliams, Thomas; Cannon, Shannon; Devidas, Meenakshi; Stork, Linda; Aplenc, Richard

2014-11-01

Sinusoidal obstruction syndrome is a complication of therapy for pediatric ALL and may be modified by thiopurine methyltransferase activity as well as by MTHFR genotype. We assessed TPMT *3A, *3B, *3C, and MTHFR C677T and A1298C germline genetic polymorphisms among 351 patients enrolled in the thioguanine treatment arm of CCG-1952 clinical trial. TPMT and MTHFR C677T genotypes were not associated with SOS risk. The combination of MTHFR and TPMT variant genotypes was not associated with SOS risk. These suggest that germline genetic variation in TPMT and MTHFR do not significantly alter SOS risk in patients exposed to thioguanine. © 2014 Wiley Periodicals, Inc.

NUDT15 and TPMT genetic polymorphisms are related to 6-mercaptopurine intolerance in children treated for acute lymphoblastic leukemia at the Children's Cancer Center of Lebanon.

PubMed

Zgheib, Nathalie K; Akika, Reem; Mahfouz, Rami; Aridi, Carol Al; Ghanem, Khaled M; Saab, Raya; Abboud, Miguel R; Tarek, Nidale; El Solh, Hassan; Muwakkit, Samar A

2017-01-01

Interindividual variability in thiopurine-related toxicity could not be completely explained by thiopurine S-methyltransferase (TPMT) polymorphisms, as a number of patients who are homozygous wild type or normal for TPMT still develop toxicity that necessitates 6-mercaptopurine (MP) dose reduction or protocol interruption. Recently, few studies reported on an inherited nucleoside diphosphate-linked moiety X motif 15 (NUDT15) c.415C>T low-function variant that is associated with decreased thiopurine metabolism and leukopenia in childhood acute lymphoblastic leukemia (ALL) and other diseases. The aim of this study is to measure the frequency of TPMT and NUDT15 polymorphisms and assess whether they are predictors of MP intolerance in children treated for ALL. One hundred thirty-seven patients with ALL of whom 121 were Lebanese were evaluated. MP dose intensity was calculated as the ratio of the tolerated MP dose to planned dose during continuation phase to maintain an absolute neutrophil count (ANC) dose above 300 per μl. One patient was NUDT15 heterozygous TC and tolerated only 33.33% of the planned MP dose, which was statistically significantly different from the median-tolerated MP dose intensity of the rest of the cohort (76.00%). Three patients had the TPMT*3A haplotype and tolerated 40.00-66.66% of the planned MP dose, which was also statistically significantly different from the rest of the cohort. This is the first report on the association of TPMT and NUDT15 polymorphisms with MP dose intolerance in Arab patients with ALL. Genotyping for additional polymorphisms may be warranted for potential gene/allele-dose effect. © 2016 Wiley Periodicals, Inc.

Current use of pharmacogenetic testing: a national survey of thiopurine methyltransferase testing prior to azathioprine prescription.

PubMed

Fargher, E A; Tricker, K; Newman, W; Elliott, R; Roberts, S A; Shaffer, J L; Bruce, I; Payne, K

2007-04-01

Azathioprine is an immunosuppressant prescribed for the treatment of inflammatory conditions and after organ transplantation. Risk of neutropaenia has limited the effective use of azathioprine (AZA) and driven requirements for careful monitoring and blood tests. Thiopurine methyltransferase (TPMT) is a genetically moderated key enzyme involved in the metabolism of AZA that can be used to stratify individuals into different levels of risk of developing neutropaenia. Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). To identify the current uptake of TPMT enzyme-level testing, TPMT genotype testing, and, the role of guidelines; to inform the prescribing and monitoring of AZA. A survey was mailed to a consultant dermatologist, gastroenterologist, and rheumatologist at every NHS Hospital Trust in England. The survey comprised mainly closed questions exploring: use of AZA and monitoring; use of TPMT enzyme-level testing and genotype testing; and, the role of guidelines to guide prescribing practice. A 70% (n=287) response rate was obtained. The majority of respondents reported prescribing AZA (99%, n=283). Prescribing and monitoring patterns differed between individual respondents and between the three disciplines. TPMT enzyme-level testing was reportedly used by 67% (n=189) of respondents, but this differed by discipline (dermatologists 94%, gastroenterologists 60%, rheumatologists 47%). In 91% of cases enzyme-level testing was carried out prior to prescribing AZA. Genotype testing is not typically available to NHS clinicians but 15 clinicians (six dermatologists, six gastroenterologists, three rheumatologists) reported using it. Most consultants (82%) reported using guidelines to inform their AZA prescribing and monitoring (dermatologists 81%, gastroenterologists 75%, rheumatologists 94%). Two-thirds of the consultants surveyed in England are using TPMT enzyme-level testing, prior to AZA treatment. Uptake differs between specialities. High uptake of TPMT enzyme-level testing by dermatologists, compared with gastroenterologists and rheumatologists, may reflect national guidelines advocating its use prior to AZA. Uptake of enzyme-level testing may alter in other specialties as other guidelines are developed.

Oral Azathioprine for Recalcitrant Pediatric Atopic Dermatitis: Clinical Response and Thiopurine Monitoring

PubMed Central

Caufield, Maura; Tom, Wynnis L.

2012-01-01

Background Azathioprine is prescribed as a corticosteroid-sparing agent for many inflammatory conditions, including refractory atopic dermatitis (AD). There is limited prospective data on its appropriate use and monitoring for children with AD. Objectives This study was designed to assess clinical response to azathioprine, determine the necessity for repeat measurement of thiopurine methyltransferase (TPMT) activity during treatment, and test the utility of measuring levels of the metabolites 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP). Methods Twelve children with severe, recalcitrant AD were treated with oral azathioprine and followed prospectively. Disease severity was determined by the SCORing Atopic Dermatitis Index. Baseline TPMT activity was measured and this was repeated along with 6-TGN and 6-MMP measurement at times of stable improvement, inadequate response, or change in response. Results Azathioprine therapy was associated with clinical improvement in all but one subject. There were few adverse effects. Three subjects showed a significant change in TPMT activity during treatment: two had a mild decrease and one demonstrated enzyme inducibility with an increase from the intermediate to the normal activity range. These changes, but not 6-TGN or 6-MMP levels, inversely correlated with the clinical response to therapy. Limitations Small sample size Conclusions Azathioprine can be of benefit in the treatment of recalcitrant pediatric AD. Repeat assessment of TPMT activity may be helpful for evaluation of non–response or change in response and warrants further study. In contrast, measurement of thiopurine metabolites during treatment was not clinically useful. PMID:22892285

Genetic polymorphism of inosine-triphosphate-pyrophosphatase influences mercaptopurine metabolism and toxicity during treatment of acute lymphoblastic leukemia individualized for thiopurine-S-methyl-transferase status.

PubMed

Stocco, Gabriele; Crews, Kristine R; Evans, William E

2010-01-01

Although genetic polymorphisms in the gene encoding human thiopurine methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity, there are many patients with wild-type TPMT who develop toxicity. Furthermore, when mercaptopurine dosages are adjusted in patients who are heterozygous at the TPMT locus, there are still some patients who develop toxicity for reasons that are not fully understood. Therefore, we recently studied the effects of a common polymorphism in another gene encoding an enzyme involved in mercaptopurine metabolism (SNP rs1127354 in inosine-triphospate-pyrophosphatase, ITPA), showing that genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of febrile neutropenia following combination chemotherapy of acute lymphoblastic leukemia (ALL) in which mercaptopurine doses are individualized based on TPMT genotype. In this review, we summarize the knowledge available about the effect and clinical relevance of TPMT and ITPA on mercaptopurine pharmacogenomics, with a particular focus on the use of this medication in pediatric patients with ALL. Reader will gain insights into: i) the effects of pharmacogenomic traits on mercaptopurine toxicity and efficacy for the treatment of ALL and ii) individualization strategies that can be used to mitigate toxicity without compromising efficacy in pediatric patients with ALL. Mercaptopurine dose can be adjusted on the basis of TPMT genotype to mitigate toxicity in pediatric patients with ALL. As treatment is individualized in this way for the most relevant genetic determinant of drug response (i.e., for mercaptopurine, TPMT), the importance of other genetic polymorphisms emerges (e.g., ITPA).

Impact of Genetic Polymorphisms on 6-Thioguanine Nucleotide Levels and Toxicity in Pediatric Patients with IBD Treated with Azathioprine.

PubMed

Lee, Mi-Na; Kang, Ben; Choi, So Yoon; Kim, Mi Jin; Woo, Sook Young; Kim, Jong-Won; Choe, Yon Ho; Lee, Soo-Youn

2015-12-01

Thiopurine-related toxicity results in discontinuation of therapy in up to 30% of patients with inflammatory bowel disease. Although thiopurine S-methyltransferase (TPMT) is implicated in toxicity, not all toxicity can be attributed to TPMT polymorphisms. We investigated the effects of polymorphisms of genes involved in thiopurine and folate metabolism pathways on 6-thioguanine nucleotide levels and toxicity. Retrospective clinical data and blood samples were collected from 132 pediatric patients with inflammatory bowel disease treated with azathioprine. Eighty-seven genetic polymorphisms of 30 genes were screened using the MassARRAY system, and 70 polymorphisms of 28 genes were selected for further analysis. TPMT genotype (P < 0.001), concurrent use of mesalazine (P = 0.006), ABCC5 (rs2293001) (P < 0.001), ITPA (rs2236206 and rs8362) (P = 0.010 and P = 0.003), and ABCB1 (rs2032582) (P = 0.028) were all associated with the ratio of 6-thioguanine nucleotides to azathioprine dose. ADK (rs10824095) (P = 0.004, odds ratio [OR] = 6.220), SLC29A1 (rs747199) (P = 0.016, OR = 5.681), and TYMS (rs34743033) (P = 0.045, OR = 3.846) were associated with neutropenia. ABCC1 (rs2074087) (P = 0.022, OR = 3.406), IMPDH1 (rs2278294) (P = 0.027, OR = 0.276), and IMPDH2 (rs11706052) (P = 0.034, OR = 3.639) had a significant impact on lymphopenia. This study describes genetic polymorphisms in genes whose products may affect pharmacokinetics and which may predict the relative likelihood of benefit or risk from thiopurine treatment. These findings may serve as a basis for personalized thiopurine therapy in pediatric patients with inflammatory bowel disease, although our data need to be validated in further studies.

A cost effectiveness analysis of thiopurine methyltransferase testing for guiding 6-mercaptopurine dosing in children with acute lymphoblastic leukemia.

PubMed

Donnan, Jennifer R; Ungar, Wendy J; Mathews, Maria; Hancock-Howard, Rebecca L; Rahman, Proton

2011-08-01

An increased understanding of the genetic basis of disease creates a demand for personalized medicine and more genetic testing for diagnosis and treatment. The objective was to assess the incremental cost-effectiveness per life-month gained of thiopurine methyltransferase (TPMT) genotyping to guide doses of 6-mercaptopurine (6-MP) in children with acute lymphoblastic leukemia (ALL) compared to enzymatic testing and standard weight-based dosing. A cost-effectiveness analysis was conducted from a health care system perspective comparing costs and consequences over 3 months. Decision analysis was used to evaluate the impact of TPMT tests on preventing myelosuppression and improving survival in ALL patients receiving 6-MP. Direct medical costs included laboratory tests, medications, physician services, pharmacy and inpatient care. Probabilities were derived from published evidence. Survival was measured in life-months. The robustness of the results to variable uncertainty was tested in one-way sensitivity analyses. Probabilistic sensitivity analysis examined the impact of parameter uncertainty and generated confidence intervals around point estimates. Neither of the testing interventions showed a benefit in survival compared to weight-based dosing. Both test strategies were more costly compared to weight-based dosing. Incremental costs per child (95% confidence interval) were $277 ($112, $442) and $298 ($392, $421) for the genotyping and phenotyping strategies, respectively, compared to weight-based dosing. The present analysis suggests that screening for TPMT mutations using either genotype or enzymatic laboratory tests prior to the administration of 6-MP in pediatric ALL patients is not cost-effective. Copyright © 2011 Wiley-Liss, Inc.

Common misconceptions about 5-aminosalicylates and thiopurines in inflammatory bowel disease

PubMed Central

Gisbert, Javier P; Chaparro, María; Gomollón, Fernando

2011-01-01

Misconceptions are common in the care of patients with inflammatory bowel disease (IBD). In this paper, we state the most commonly found misconceptions in clinical practice and deal with the use of 5-aminosalicylates and thiopurines, to review the related scientific evidence, and make appropriate recommendations. Prevention of errors needs knowledge to avoid making such errors through ignorance. However, the amount of knowledge is increasing so quickly that one new danger is an overabundance of information. IBD is a model of a very complex disease and our goal with this review is to summarize the key evidence for the most common daily clinical problems. With regard to the use of 5-aminosalicylates, the best practice may to be consider abandoning the use of these drugs in patients with small bowel Crohn’ s disease. The combined approach with oral plus topical 5-aminosalicylates should be the first-line therapy in patients with active ulcerative colitis; once-daily treatment should be offered as a first choice regimen due to its better compliance and higher efficacy. With regard to thiopurines, they seem to be as effective in ulcerative colitis as in Crohn’ s disease. Underdosing of thiopurines is a form of undertreatment. Thiopurines should probably be continued indefinitely because their withdrawal is associated with a high risk of relapse. Mercaptopurine is a safe alternative in patients with digestive intolerance or hepatotoxicity due to azathioprine. Finally, thiopurine methyltransferase (TPMT) screening cannot substitute for regular monitoring because the majority of cases of myelotoxicity are not TPMT-related. PMID:21941413

Two brothers with skewed thiopurine metabolism in ulcerative colitis treated successfully with allopurinol and mercaptopurine dose reduction.

PubMed

Hoentjen, Frank; Hanauer, Stephen B; de Boer, Nanne K; Rubin, David T

2012-01-01

Thiopurine therapy effectively maintains remission in inflammatory bowel disease. However, many patients are unable to achieve optimum benefits from azathioprine or 6-mercaptopurine because of undesirable metabolism related to high thiopurine methyltransferase (TPMT) activity characterized by hepatic transaminitis secondary to increased 6-methylmercaptopurine (6-MMP) production and reduced levels of therapeutic 6-thioguanine nucleotide (6-TGN). Allopurinol can optimize this skewed metabolism. We discuss two brothers who were both diagnosed with ulcerative colitis (UC). Their disease remained active despite oral and topical mesalamines. Steroids followed by 6-mercaptopurine (MP) were unsuccessfully introduced for both patients and both were found to have high 6-MMP and low 6-TGN levels, despite normal TMPT enzyme activity, accompanied by transaminitis. Allopurinol was introduced in combination with MP dose reduction. For both brothers addition of allopurinol was associated with successful remission and optimized MP metabolites. These siblings with active UC illustrate that skewed thiopurine metabolism may occur despite normal TPMT enzyme activity and can lead to adverse events in the absence of disease control. We confirm previous data showing that addition of allopurinol can reverse this skewed metabolism, and reduce both hepatotoxicity and disease activity, but we now also introduce the concept of a family history of preferential MP metabolism as a clue to effective management for other family members.

Myelotoxicity after high-dose methotrexate in childhood acute leukemia is influenced by 6-mercaptopurine dosing but not by intermediate thiopurine methyltransferase activity

PubMed Central

Levinsen, Mette; Rosthøj, Susanne; Nygaard, Ulrikka; Heldrup, Jesper; Harila-Saari, Arja; Jonsson, Olafur G.; Bechensteen, Anne Grete; Abrahamsson, Jonas; Lausen, Birgitte; Frandsen, Thomas L.; Weinshilboum, Richard M.; Schmiegelow, Kjeld

2015-01-01

Purpose Through enhancement of 6-mercaptopurine (6MP) bioavailability and inhibition of purine de novo synthesis high-dose methotrexate (HD-MTX) may increase incorporation into DNA of 6-thioguanine nucleotides (6TGN), the cytotoxic metabolites of 6MP. Patients with intermediate activity of thiopurine methyltransferase (TPMTIA) have higher cytosol 6-thioguanine nucleotide levels. We investigated toxicity following HD-MTX during MTX/6MP maintenance therapy in relation to 6MP and TPMT. Methods Using linear mixed models, we explored myelo- and hepatotoxicity in relation to 6MP dosage and TPMT phenotype following 1,749 HD-MTX courses to 411 children with acute lymphoblastic leukemia on maintenance therapy. Results The degree of myelosuppression following HD-MTX was similar for patients with TPMTIA and patients with high TPMT activity (TPMTHA), when HD-MTX started with same blood counts and 6MP doses. However, since TPMTIA had lower blood counts at initiation of HD-MTX compared to TPMTHA patients (median WBC 2.8 vs. 3.3 ×109/L, P=0.01; median ANC 1.4 vs. 1.7 ×109/L, P=0.02), TPMTIA continued to have lower WBC and ANC levels compared to TPMTHA during all 28 days after HD-MTX (relative difference: 9% (95% CI: 2-17%), P=0.02 and 21% (95% CI: 6-39%), P=0.005). Still, the fractional decrease in WBC and ANC levels after HD-MTX did not differ between TPMTIA and TPMTHA patients (P=0.47 and P=0.38). The degree of leukopenia, neutropenia, thrombocytopenia and rise in aminotransferases were all significantly related to 6MP dose (P<0.001 for all analyses). Conclusion For both TPMTIA and TPMTHA patients dose of 6MP prior to HD-MTX should be guided by pre-HD-MTX blood counts, but not by TPMT activity. PMID:25347948

Usefulness of salicylate and thiopurine coprescription in steroid-dependent ulcerative colitis and withdrawal strategies

PubMed Central

Bermejo, Fernando; Gisbert, Javier P.

2010-01-01

5-aminosalicylic acid (5-ASA) and thiopurines (azathioprine and mercaptopurine) are the most common drugs used as a maintenance treatment for ulcerative colitis. A considerable proportion of these patients develop corticosteroid dependency, and thiopurines are the standard treatment in this scenario. Dual prescriptions of both thiopurines and 5-ASA are common practice in steroid-dependent ulcerative colitis, in an attempt to optimize the efficacy of therapy. On the one hand, the potential protective role of 5-ASA against colorectal cancer argues in favour of prescription of both medications. The possible synergism between the two drugs, because of the inhibition of thiopurine methyltransferase (TPMT) enzyme activity by 5-ASA, has been postulated as another justification for dual prescription. However, existing evidence does not support that this combined strategy is superior to monotherapy with thiopurines. On the other hand, in patients showing prolonged disease remission, the possibility of discontinuing maintenance treatment can be considered on an individualized basis. The high frequency of relapses after thiopurine withdrawal should always be taken into account, but the potential adverse effects of the medication also need to be considered. A properly indicated treatment with thiopurines may need to be continued for life in many patients. PMID:23251733

Prospective Evaluation of Pharmacogenomics and Metabolite Measurements upon Azathioprine Therapy in Inflammatory Bowel Disease: An Observational Study.

PubMed

Fangbin, Zhang; Xiang, Gao; Liang, Ding; Hui, Liu; Xueding, Wang; Baili, Chen; Huichang, Bi; Yinglian, Xiao; Peng, Cheng; Lizi, Zhao; Yanjun, Chu; Feng, Xu; Minhu, Chen; Min, Huang; Pinjin, Hu

2016-04-01

Up to approximately 40% to 50% of patients discontinue thiopurine therapy during the course of inflammatory bowel disease (IBD). We investigated the role of the metabolite thiopurine in IBD treatment. This was a prospective study. IBD patients receiving azathioprine (AZA) were prospectively included. Thiopurine methyltransferase (TPMT) genotypes were examined before therapy, and thiopurine metabolite levels were examined at weeks 2, 4, 8, 12, 24, and 48. In total, 132 patients were included. The frequency of leucopenia increased at 6-thioguanine nucleotide (6-TGN) levels ≥420  pmol/8 × 10(8) RBC (odds ratio [OR] = 7.9; 95% confidence interval (95%CI): 3.5-18.0; P < 0.001) and increased more during the initial 12 weeks of thiopurine therapy (OR = 16.0; 95%CI: 5.7-44.9; P < 0.001). The patients with 6-TGN levels ≥420 pmol/8 × 10 RBC at weeks 4, 8, and 12 had an increased likelihood of leucopenia. Clinical response increased at 6-TGN levels ≥225 pmol/8 × 10(8) RBC (OR = 13.5; 95% CI: 3.7-48.9; P < 0.001) in Crohn disease (CD) patients. The CD patients with 6-TGN levels ≥225 pmol/8 × 10(8) RBC at weeks 8, 12, and 24 had an increased likelihood of successful clinical response. TPMT*3C had a specificity of 100%, but a sensitivity of 8% for predicting leucopenia.A 6-TGN level between 225 and 420 pmol/8 × 10(8) RBC could be a therapeutic window in patients receiving AZA therapy, and it could likely predict leucopenia in the initial 12 weeks of AZA therapy and a reasonable chance of successful clinical response in CD patients. The value of TPMT genotyping before thiopurine therapy is limited in Chinese patients with IBD, considering the low sensitivity of predicting leucopenia.

Multilocus Genotypes of Relevance for Drug Metabolizing Enzymes and Therapy with Thiopurines in Patients with Acute Lymphoblastic Leukemia

PubMed Central

Stocco, Gabriele; Franca, Raffaella; Verzegnassi, Federico; Londero, Margherita; Rabusin, Marco; Decorti, Giuliana

2013-01-01

Multilocus genotypes have been shown to be of relevance for using pharmacogenomic principles to individualize drug therapy. As it relates to thiopurine therapy, genetic polymorphisms of TPMT are strongly associated with the pharmacokinetics and clinical effects of thiopurines (mercaptopurine and azathioprine), influencing their toxicity and efficacy. We have recently demonstrated that TPMT and ITPA genotypes constitute a multilocus genotype of pharmacogenetic relevance for children with acute lymphoblastic leukemia (ALL) receiving thiopurine therapy. The use of high-throughput genomic analysis allows identification of additional candidate genetic factors associated with pharmacogenetic phenotypes, such as TPMT enzymatic activity: PACSIN2 polymorphisms have been identified by a genome-wide analysis, combining evaluation of polymorphisms and gene expression, as a significant determinant of TPMT activity in the HapMap CEU cell lines and the effects of PACSIN2 on TPMT activity and mercaptopurine induced adverse effects were confirmed in children with ALL. Combination of genetic factors of relevance for thiopurine metabolizing enzyme activity, based on the growing understanding of their association with drug metabolism and efficacy, is particularly promising for patients with pediatric ALL. The knowledge basis and clinical applications for multilocus genotypes of importance for therapy with mercaptopurine in pediatric ALL is discussed in the present review. PMID:23335936

NUDT15 gene polymorphism related to mercaptopurine intolerance in Taiwan Chinese children with acute lymphoblastic leukemia.

PubMed

Liang, D-C; Yang, C-P; Liu, H-C; Jaing, T-H; Chen, S-H; Hung, I-J; Yeh, T-C; Lin, T-H; Lai, C-L; Lai, C-Y; Shih, L-Y

2016-11-01

A recent study identified a variant of the NUDT15 gene (rs116855232 C>T) associated with intolerance to thiopurine in Korean patients with Crohn's disease. This study prompted us to substantiate the finding in a Taiwanese population. Four hundred and four children with acute lymphoblastic leukemia (ALL), and 100 adults with chronic immune thrombocytopenic purpura or localized lymphoma having normal bone marrow were examined. Two candidate gene approaches, pyrosequencing for NUDT15 and TaqMan assay for thiopurine methyltransferase (TPMT) genotyping (rs1142345 A>G), were performed. We showed a risk allele frequency of NUDT15 of 11.6% in children with ALL and 15.5% in adults. By contrast, the risk allele frequency of TPMT was only 1.6% in children with ALL and 0.5% in adults. The high frequency of risk variant for NUDT15, but not the very low frequency of risk variant for TPMT, was closely associated with the intolerance to mercaptopurine in children with ALL in Taiwan, contrast to that of European descent. In regard to NUDT15 polymorphism, the maximal tolerable daily doses of mercaptopurine in homozygotes, heterozygotes and wild-type groups were 9.4 mg m -2 , 30.7 mg m -2 and 44.1 mg m -2 , respectively. The outcomes did not differ significantly among the different genotypes.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity.

PubMed

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R; Thierfelder, William E; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W; Ramsey, Laura B; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S; Pui, Ching-Hon; Relling, Mary V; Evans, William E

2012-11-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

PubMed Central

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R.; Thierfelder, William E.; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W.; Ramsey, Laura B.; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S.; Pui, Ching-Hon; Relling, Mary V.; Evans, William E.

2012-01-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy. PMID:22846425

Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy

PubMed Central

Adam de Beaumais, Tiphaine; Fakhoury, May; Medard, Yves; Azougagh, Said; Zhang, Daolun; Yakouben, Karima; Jacqz-Aigrain, Evelyne

2011-01-01

AIMS 6-mercaptopurine (6-MP) is used in the treatment of childhood acute lymphoblastic leukaemia (ALL). Its red blood cell (RBC) metabolite concentrations (6-thioguanine [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) are related to drug response. We investigated the impact of non-genetic covariates and pharmacogenetic polymorphisms affecting thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) on 6-MP metabolism and response. METHODS Sixty-six children with ALL treated according to EORTC 58951 protocol were included in this study. Six patients had a heterozygous genotype for the most common TPMT polymorphisms, nine for ITPA 94 C > A and 17 for ITPA IVS2+21 A > C. 6-MP metabolites concentrations were analyzed by mixed model analysis. RESULTS During maintenance, steady-state RBC 6-TGN concentrations were lower in patients aged 6 years or younger (493 pmol/8 × 108RBC) than in older children (600 pmol/8 × 108RBC). 6-MMPN concentrations were low in patients with TPMT variant/wild-type ITPA (1862 pmol/8 × 108RBC), intermediate in wild-type patients and high (16468 pmol/8 × 108RBC) in patients wild-type TPMT/variant ITPA. A 6-MMPN threshold of 5000 pmol/8 × 108RBC was associated with an increased risk of hepatotoxicity. CONCLUSION In this study, age and both TPMT and ITPA genotypes influenced 6-MP metabolism. High 6-MMPN was associated with hepatotoxicity. These pharmacological tools should be used to monitor ALL treatment in children. PMID:21395650

Pharmacogenetics of anti-cancer drugs: State of the art and implementation - recommendations of the French National Network of Pharmacogenetics.

PubMed

Quaranta, Sylvie; Thomas, Fabienne

2017-04-01

Individualized treatment is of special importance in oncology because the drugs used for chemotherapy have a very narrow therapeutic index. Pharmacogenetics may contribute substantially to clinical routine for optimizing cancer treatment to limit toxic effects while maintaining efficacy. This review presents the usefulness of pharmacogenetic tests for some key applications: dihydropyrimidine dehydrogenase (DPYD) genotyping for fluoropyrimidine (5-fluorouracil, capecitabine), UDP glucuronosylstransferase (UGT1A1) for irinotecan and thiopurine S-methyltransferase (TPMT) for thiopurine drugs. Depending on the level of evidence, the French National Network of Pharmacogenetics (RNPGx) has issued three levels of recommendations for these pharmacogenetic tests: essential, advisable, and potentially useful. Other applications, for which the level of evidence is still discussed, will be evoked in the final section of this review. Copyright © 2017 Société française de pharmacologie et de thérapeutique. Published by Elsevier Masson SAS. All rights reserved.

Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy.

PubMed

Adam de Beaumais, Tiphaine; Fakhoury, May; Medard, Yves; Azougagh, Said; Zhang, Daolun; Yakouben, Karima; Jacqz-Aigrain, Evelyne

2011-04-01

6-mercaptopurine (6-MP) is used in the treatment of childhood acute lymphoblastic leukaemia (ALL). Its red blood cell (RBC) metabolite concentrations (6-thioguanine [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) are related to drug response. We investigated the impact of non-genetic covariates and pharmacogenetic polymorphisms affecting thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) on 6-MP metabolism and response. Sixty-six children with ALL treated according to EORTC 58951 protocol were included in this study. Six patients had a heterozygous genotype for the most common TPMT polymorphisms, nine for ITPA 94 C > A and 17 for ITPA IVS2+21 A > C. 6-MP metabolites concentrations were analyzed by mixed model analysis. During maintenance, steady-state RBC 6-TGN concentrations were lower in patients aged 6 years or younger (493 pmol/8 × 10(8) RBC) than in older children (600 pmol/8 × 10(8) RBC). 6-MMPN concentrations were low in patients with TPMT variant/wild-type ITPA (1862 pmol/8 × 10(8) RBC), intermediate in wild-type patients and high (16468 pmol/8 × 10(8) RBC) in patients wild-type TPMT/variant ITPA. A 6-MMPN threshold of 5000 pmol/8 × 10(8) RBC was associated with an increased risk of hepatotoxicity. In this study, age and both TPMT and ITPA genotypes influenced 6-MP metabolism. High 6-MMPN was associated with hepatotoxicity. These pharmacological tools should be used to monitor ALL treatment in children. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

Pharmacogenetic determinants of mercaptopurine disposition in children with acute lymphoblastic leukemia.

PubMed

Adam de Beaumais, Tiphaine; Jacqz-Aigrain, Evelyne

2012-09-01

The backbone of drug therapy used in acute lymphoblastic leukemia (ALL) in children includes 6-mercaptopurine (6-MP). Intracellular metabolism of this prodrug is a key component of the therapeutic response. Many metabolizing enzymes are involved in 6-MP disposition and active 6-MP metabolites are represented by 6-thioguanine nucleotides (6-TGN) and methylated metabolites primarily methylated by the thiopurine S-methyltransferase enzyme (TPMT). The genetic polymorphism affecting TPMT activity displays an important inter-subject variability in metabolites pharmacokinetics and influences the balance between 6-MP efficacy and toxicity: patients with high 6-TGN levels are at risk of myelosuppression while patients with high levels of methylated derivates are at hepatotoxic risk. However, the genetic TPMT polymorphism does not explain all 6-MP adverse events and some severe toxicities leading to life-threatening conditions remain unexplained. Additional single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in 6-MP metabolism and 6-MP transporters may also be responsible for this inter-individual 6-MP response variability. This review presents the pharmacogenetic aspects of 6-MP metabolism in great detail. We have focused on published data on ALL treatment supporting the great potential of 6-MP pharmacogenetics to improve efficacy, tolerance, and event-free survival rates in children with ALL.

Methotrexate/6-mercaptopurine maintenance therapy influences the risk of a second malignant neoplasm after childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study.

PubMed

Schmiegelow, Kjeld; Al-Modhwahi, Ibrahim; Andersen, Mette Klarskov; Behrendtz, Mikael; Forestier, Erik; Hasle, Henrik; Heyman, Mats; Kristinsson, Jon; Nersting, Jacob; Nygaard, Randi; Svendsen, Anne Louise; Vettenranta, Kim; Weinshilboum, Richard

2009-06-11

Among 1614 children with acute lymphoblastic leukemia (ALL) treated with the Nordic Society for Paediatric Haematology and Oncology (NOPHO) ALL-92 protocol, 20 patients developed a second malignant neoplasm (SMN) with a cumulative risk of 1.6% at 12 years from the diagnosis of ALL. Nine of the 16 acute myeloid leukemias or myelodysplastic syndromes had monosomy 7 (n = 7) or 7q deletions (n = 2). In Cox multivariate analysis, longer duration of oral 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy (P = .02; longest for standard-risk patients) and presence of high hyperdiploidy (P = .07) were related to increased risk of SMN. Thiopurine methyltransferase (TPMT) methylates 6MP and its metabolites, and thus reduces cellular levels of cytotoxic 6-thioguanine nucleotides. Of 524 patients who had erythrocyte TPMT activity measured, the median TPMT activity in 9 patients developing an SMN was significantly lower than in the 515 that did not develop an SMN (median, 12.1 vs 18.1 IU/mL; P = .02). Among 427 TPMT wild-type patients for whom the 6MP dose was registered, those who developed SMN received higher average 6MP doses than the remaining patients (69.7 vs 60.4 mg/m2; P = .03). This study indicates that the duration and intensity of 6MP/MTX maintenance therapy of childhood ALL may influence the risk of SMNs in childhood ALL.

The multifunctional application of microfluidic lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) within the field of bioanalytics

NASA Astrophysics Data System (ADS)

März, Anne; Mönch, Bettina; Walter, Angela; Bocklitz, Thomas; Schumacher, Wilm; Rösch, Petra; Kiehntopf, Michael; Popp, Jürgen

2011-07-01

This contribution will present a variety of applications of lab-on-a-chip surface enhanced Raman spectroscopy in the field of bioanalytic. Beside the quantification and online monitoring of drugs and pharmaceuticals, determination of enzyme activity and discrimination of bacteria are successfully carried out utilizing LOC-SERS. The online-monitoring of drugs using SERS in a microfluidic device is demonstrated for nicotine. The enzyme activity of thiopurine methyltransferase (TPMT) in lysed red blood cells is determined by SERS in a lab-on-a-chip device. To analyse the activity of TPMT the metabolism of 6-mercaptopurine to 6-methylmercaptopurine is investigated. The discrimination of bacteria on strain level is carried out with different E. coli strains. For the investigations, the bacteria are busted by ultra sonic to achieve a high information output. This sample preparation provides the possibility to detect SERS spectra containing information of the bacterial cell walls as well as of the cytoplasm. This contribution demonstrates the great potential of LOC-SERS in the field of bioanalytics.

Fast and slow methylators: do racial differences influence risk of allograft rejection?

PubMed

Chocair, P R; Duley, J A; Sabbaga, E; Arap, S; Simmonds, H A; Cameron, J S

1993-06-01

A catabolic route for azathioprine involving methylation by thiopurine methyltransferase has been directly implicated in the drug's immunosuppressive efficacy. Since ethnic differences in thiopurine methyltransferase activity have been reported in a study of Lapps, this study compared the distribution of thiopurine methyltransferase activity in erythrocyte lysates from 134 healthy, randomly selected subjects living in Brazil, comprising 39 blacks (i.e. Afro-Brazilians), 33 white subjects, 30 mixed-race subjects, and 32 Brazilian-residing Japanese subjects. The results demonstrated bimodality of thiopurine methyltransferase activity compatible with genetic polymorphism in the white, black and mixed-race groups, but not in the Japanese, who were homogeneously 'fast methylators' (high thiopurine methyltransferase activity). Thiopurine methyltransferase activity was generally higher in Brazilian males than females, and some individuals in the black and mixed-race groups had very high activity. Azathioprine-immunosuppressed transplant patients with thiopurine methyltransferase activity above 35 pmol/h/mgHb have previously been shown to have significantly poorer outcomes. Using this thiopurine methyltransferase value as the cut-off point between 'poor responders' and 'good responders' to azathioprine, 65% of the Japanese, 59% of the black subjects, and 63% of the mixed-race subjects fell into the 'poor responder' category, compared with only 42% of the white group. Interestingly, this approximately 20% difference in azathioprine response corresponds to the racial differences seen in allograft survival.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia

PubMed Central

Hawwa, Ahmed F; Collier, Paul S; Millership, Jeff S; McCarthy, Anthony; Dempsey, Sid; Cairns, Carole; McElnay, James C

2008-01-01

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECTThe cytotoxic effects of 6-mercaptopurine (6-MP) were found to be due to drug-derived intracellular metabolites (mainly 6-thioguanine nucleotides and to some extent 6-methylmercaptopurine nucleotides) rather than the drug itself.Current empirical dosing methods for oral 6-MP result in highly variable drug and metabolite concentrations and hence variability in treatment outcome. WHAT THIS STUDY ADDSThe first population pharmacokinetic model has been developed for 6-MP active metabolites in paediatric patients with acute lymphoblastic leukaemia and the potential demographic and genetically controlled factors that could lead to interpatient pharmacokinetic variability among this population have been assessed.The model shows a large reduction in interindividual variability of pharmacokinetic parameters when body surface area and thiopurine methyltransferase polymorphism are incorporated into the model as covariates.The developed model offers a more rational dosing approach for 6-MP than the traditional empirical method (based on body surface area) through combining it with pharmacogenetically guided dosing based on thiopurine methyltransferase genotype. AIMS To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. METHODS Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m−2 day−1). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. RESULTS The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model. CONCLUSIONS The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype. PMID:18823306

Therapeutic drug monitoring of antimetabolic cytotoxic drugs

PubMed Central

Lennard, L

1999-01-01

Therapeutic drug monitoring is not routinely used for cytotoxic agents. There are several reasons, but one major drawback is the lack of established therapeutic concentration ranges. Combination chemotherapy makes the establishment of therapeutic ranges for individual drugs difficult, the concentration-effect relationship for a single drug may not be the same as that when the drug is used in a drug combination. Pharmacokinetic optimization protocols for many classes of cytotoxic compounds exist in specialized centres, and some of these protocols are now part of large multicentre trials. Nonetheless, methotrexate is the only agent which is routinely monitored in most treatment centres. An additional factor, especially in antimetabolite therapy, is the existence of pharmacogenetic enzymes which play a major role in drug metabolism. Monitoring of therapy could include assay of phenotypic enzyme activities or genotype in addition to, or instead of, the more traditional measurement of parent drug or drug metabolites. The cytotoxic activities of mercaptopurine and fluorouracil are regulated by thiopurine methyltransferase (TPMT) and dihydropyrimidine dehydrogenase (DPD), respectively. Lack of TPMT functional activity produces life-threatening mercaptopurine myelotoxicity. Very low DPD activity reduces fluorouracil breakdown producing severe cytotoxicity. These pharmacogenetic enzymes can influence the bioavailability, pharmacokinetics, toxicity and efficacy of their substrate drugs. PMID:10190647

NUDT15 R139C Variants Increase the Risk of Azathioprine-Induced Leukopenia in Chinese Autoimmune Patients

PubMed Central

Fei, Xiang; Shu, Qing; Zhu, Huaijun; Hua, Bingzhu; Wang, Shiying; Guo, Ling; Fang, Yun; Ge, Weihong

2018-01-01

The aim of this study was to investigate the influence of NUDT15 R139C, thiopurine S-methyltransferase (TPMT), and 6-TGN on azathioprine (AZA) induced leukopenia in Chinese autoimmune patients. Among 87 enrolled patients, 23 (26.4%) had leukopenia. The NUDT15 R139C variant was associated with leukopenia (p = 1.86 × 10−7; OR: 7.59; 95% CI: 3.16–18.21). However, TPMT genotype was not shown to be correlated with the incidence of leukopenia (p = 0.95). There was no significant difference of 6-TGN concentration between patients with or without leukopenia (p = 0.15) and no association was found in patients with NUDT15 R139C variants alleles (p = 0.62). Finally, we found that the range of 6-TGN concentrations in autoimmune diseases was much lower than the established 6-TGN monitoring range for inflammatory bowel diseases. Therefore, the variant of NUDT15 R139C is strongly associated with AZA-induced leukopenia in Chinese patients with various autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome, etc. PMID:29867468

Body Mass Index and Smoking Affect Thioguanine Nucleotide Levels in Inflammatory Bowel Disease.

PubMed

Poon, Shi Sum; Asher, Rebecca; Jackson, Richard; Kneebone, Andrew; Collins, Paul; Probert, Chris; Dibb, Martyn; Subramanian, Sreedhar

2015-08-01

Optimal levels of the thiopurine metabolite, 6-thioguanine nucleotides [6-TGN] correlate with remission of inflammatory bowel disease [IBD]. Apart from variations in the thiopurine methyl transferase [TPMT] gene, little is known about other predictors of 6-TGN levels. Obesity adversely affects response to infliximab and adalimumab and clinical course in IBD, but little is known about the interaction of thiopurines and obesity. We investigated the relationship between body mass index [BMI] and 6-TGN levels and sought to examine other predictors of 6-TGN levels. This retrospective cohort study included patients with concurrent measurements of 6-TGN and BMI. The association between 6-TGN and clinical variables including BMI was estimated using a multivariable linear regression model. Of 132 observations, 77 [58%] had Crohn's disease and 55 [42%] ulcerative colitis. BMI, smoking, and TPMT levels were associated with 6-TGN levels in multivariable analysis. Every 5kg/m(2) increase in BMI was associated with an 8% decrease in 6-TGN (0.92; 95% confidence interval [CI] 0.87-0.98; p = 0.009). Smokers had higher 6-TGN levels in comparison with non-/ex-smokers [1.43; 95% CI 1.02-2.02; p = 0.041]. Patients with intermediate TPMT had higher 6-TGN compared to those with normal levels [2.13; 95% CI 1.62-2.80; p < 0.001]. Obese patients were more likely to have sub-therapeutic 6-TGN levels and a higher methyl mercaptopurine nucleotide [MMPN/TGN] ratio despite a similar dose of thiopurines. Active smoking and intermediate TPMT values were associated with higher 6-TGN levels but increasing BMI resulted in lower 6-TGN and higher MMPN levels. This may explain the worse outcome that has been reported previously in obese IBD subjects. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Is 6-thioguanine more appropriate than 6-mercaptopurine for children with acute lymphoblastic leukaemia?

PubMed Central

Lennard, L.; Davies, H. A.; Lilleyman, J. S.

1993-01-01

The cytotoxic activity of 6-mercaptopurine (6-MP) is affected by thiopurine methyltransferase (TPMT), a genetically regulated and variable intracellular enzyme. 6-Thioguanine (6-TG), a closely related thiopurine, is less affected by that enzyme and so it may be a more reliable drug-at least for patients with constitutionally high TPMT activity. We attempted to assess its suitability as an alternative by comparing the pharmacokinetics of both drugs in a small group of children with lymphoblastic leukaemia (ALL). Patients were included who were in their second or subsequent remission, who would otherwise have received 6-MP, and on whom pharmacokinetic data concerning 6-MP metabolism had been collected in a previous remission. Plasma 6-TG concentrations were assayed following an oral dose of 40 mg m-2, and the accumulation and fluctuation of intracellular (erythrocyte, RBC) 6-TG nucleotides (6-TGNs) were measured at regular intervals during daily oral therapy. Seven children were studied. Plasma 6-TG concentrations were low and cleared within 6 h of oral dosing. At 7 days, 6-TGN concentrations ranged from 959 to 2361 pmol 8 x 10(-8) RBCs, in all cases significantly higher (P = 0.002) than those produced by the same patients on 6-MP. After a total therapy time of 35 patient months, a modest rise of alanine aminotransferase was seen on one occasion, otherwise no toxicity apart from myelosuppression was encountered. In the context used, 6-TG appears well tolerated and produces higher concentrations of intracellular cytotoxic metabolites than 6-MP. For children constitutionally 'resistant' to the traditional drug, if not all, it may be a preferable alternative. PMID:8318412

Genetic Polymorphism of Inosine Triphosphate Pyrophosphatase Is a Determinant of Mercaptopurine Metabolism and Toxicity During Treatment for Acute Lymphoblastic Leukemia

PubMed Central

Stocco, G; Cheok, MH; Crews, KR; Dervieux, T; French, D; Pei, D; Yang, W; Cheng, C; Pui, C-H; Relling, MV; Evans, WE

2009-01-01

The influence of genetic polymorphism in inosine triphosphate pyrophosphatase (ITPA) on thiopurine-induced adverse events has not been investigated in the context of combination chemotherapy for acute lymphoblastic leukemia (ALL). This study investigated the effects of a common ITPA variant allele (rs41320251) on mercaptopurine metabolism and toxicity during treatment of children with ALL. Significantly higher concentrations of methyl mercaptopurine nucleotides were found in patients with the nonfunctional ITPA allele. Moreover, there was a significantly higher probability of severe febrile neutropenia in patients with a variant ITPA allele among patients whose dose of mercaptopurine had been adjusted for TPMT genotype. In a cohort of patients whose mercaptopurine dose was not adjusted for TPMT phenotype, the TPMT genotype had a greater effect than the ITPA genotype. In conclusion, genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of severe febrile neutropenia, after combination chemotherapy for ALL in which mercaptopurine doses are individualized on the basis of TPMT genotype. PMID:18685564

Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia.

PubMed

Stocco, G; Cheok, M H; Crews, K R; Dervieux, T; French, D; Pei, D; Yang, W; Cheng, C; Pui, C-H; Relling, M V; Evans, W E

2009-02-01

The influence of genetic polymorphism in inosine triphosphate pyrophosphatase (ITPA) on thiopurine-induced adverse events has not been investigated in the context of combination chemotherapy for acute lymphoblastic leukemia (ALL). This study investigated the effects of a common ITPA variant allele (rs41320251) on mercaptopurine metabolism and toxicity during treatment of children with ALL. Significantly higher concentrations of methyl mercaptopurine nucleotides were found in patients with the nonfunctional ITPA allele. Moreover, there was a significantly higher probability of severe febrile neutropenia in patients with a variant ITPA allele among patients whose dose of mercaptopurine had been adjusted for TPMT genotype. In a cohort of patients whose mercaptopurine dose was not adjusted for TPMT phenotype, the TPMT genotype had a greater effect than the ITPA genotype. In conclusion, genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of severe febrile neutropenia, after combination chemotherapy for ALL in which mercaptopurine doses are individualized on the basis of TPMT genotype.

NUDT15 Polymorphisms Alter Thiopurine Metabolism and Hematopoietic Toxicity

PubMed Central

Moriyama, Takaya; Nishii, Rina; Perez-Andreu, Virginia; Yang, Wenjian; Klussmann, Federico Antillon; Zhao, Xujie; Lin, Ting-Nien; Hoshitsuki, Keito; Nersting, Jacob; Kihira, Kentaro; Hofmann, Ute; Komada, Yoshihiro; Kato, Motohiro; McCorkle, Robert; Li, Lie; Koh, Katsuyoshi; Najera, Cesar Rolando; Kham, Shirley Kow-Yin; Isobe, Tomoya; Chen, Zhiwei; Chiew, Edwynn Kean-Hui; Bhojwani, Deepa; Jeffries, Cynthia; Lu, Yan; Schwab, Matthias; Inaba, Hiroto; Pui, Ching-Hon; Relling, Mary V.; Manabe, Atsushi; Hori, Hiroki; Schmiegelow, Kjeld; Yeoh, Allen E. J.; Evans, William E.; Yang, Jun J.

2016-01-01

Widely used as anti-cancer and immunosuppressive agents, thiopurines have narrow therapeutic indices due to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and its clinical implications remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore, and Japan, we identified 4 NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile, p.Val18_Val19insGlyVal) that resulted in 74.4%–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across three cohorts (P=0.021, 2.1×10−5, and 0.0054, respectively; meta-analysis P=4.45×10−8, allelic effect size=−11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased its cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive thiopurine active metabolites and toxicity. Taken together, our results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy. PMID:26878724

Comparative pharmacogenetic analysis of risk polymorphisms in Caucasian and Vietnamese children with acute lymphoblastic leukemia: prediction of therapeutic outcome?

PubMed

Hoang, Phuong Thu Vu; Ambroise, Jérôme; Dekairelle, Anne-France; Durant, Jean-François; Butoescu, Valentina; Chi, Vu Luan Dang; Huynh, Nghia; Nguyen, Tan Binh; Robert, Annie; Vermylen, Christiane; Gala, Jean-Luc

2015-03-01

Acute lymphoblastic leukemia (ALL) is the most common of all paediatric cancers. Aside from predisposing to ALL, polymorphisms could also be associated with poor outcome. Indeed, genetic variations involved in drug metabolism could, at least partially, be responsible for heterogeneous responses to standardized leukemia treatments, hence requiring more personalized therapy. The aims of this study were to (a) to determine the prevalence of seven common genetic polymorphisms including those that affect the folate and/or thiopurine metabolic pathways, i.e. cyclin D1 (CCND1-G870A), γ-glutamyl hydrolase (GGH-C452T), methylenetetrahydrofolate reductase (MTHFR-C677T and MTHFR-A1298C), thymidylate synthase promoter (TYMS-TSER), thiopurine methyltransferase (TPMT*3A and TPMT*3C) and inosine triphosphate pyrophosphatase (ITPA-C94A), in Caucasian (n = 94, age < 20) and Vietnamese (n = 141, age < 16 years) childhood ALL and (b) to assess the impact of a multilocus genetic risk score (MGRS) on relapse-free survival (RFS) using a Cox proportional-hazards regression model. The prevalence of MTHFR-677TT genotype was significantly higher in Caucasians (P = 0.008), in contrast to the prevalence of TYMS-TSER*3R/3R and ITPA-94AA/AC genotypes which were significantly higher in Vietnamese (P < 0.001 and P = 0.02, respectively). Compared with children with a low MGRS (≤ 3), those with a high MGRS (≥ 4) were 2.06 (95% CI = 1.01, 4.22; P = 0.04) times more likely to relapse. Adding MGRS into a multivariate Cox regression model with race/ethnicity and four clinical variables improved the predictive accuracy of the model (AUC from 0.682 to 0.709 at 24 months). Including MGRS into a clinical model improved the predictive accuracy of short and medium term prognosis, hence confirming the association between well determined pharmacogenotypes and outcome of paediatric ALL. Whether variants on other genes associated with folate metabolism can substantially improve the predictive value of current MGRS is not known but deserves further evaluation. © 2014 The British Pharmacological Society.

Comparative pharmacogenetic analysis of risk polymorphisms in Caucasian and Vietnamese children with acute lymphoblastic leukemia: prediction of therapeutic outcome?

PubMed Central

Vu Hoang, Phuong Thu; Ambroise, Jérôme; Dekairelle, Anne-France; Durant, Jean-François; Butoescu, Valentina; Dang Chi, Vu Luan; Huynh, Nghia; Nguyen, Tan Binh; Robert, Annie; Vermylen, Christiane; Gala, Jean-Luc

2015-01-01

Aims Acute lymphoblastic leukemia (ALL) is the most common of all paediatric cancers. Aside from predisposing to ALL, polymorphisms could also be associated with poor outcome. Indeed, genetic variations involved in drug metabolism could, at least partially, be responsible for heterogeneous responses to standardized leukemia treatments, hence requiring more personalized therapy. The aims of this study were to (a) to determine the prevalence of seven common genetic polymorphisms including those that affect the folate and/or thiopurine metabolic pathways, i.e. cyclin D1 (CCND1-G870A), γ-glutamyl hydrolase (GGH-C452T), methylenetetrahydrofolate reductase (MTHFR-C677T and MTHFR-A1298C), thymidylate synthase promoter (TYMS-TSER), thiopurine methyltransferase (TPMT*3A and TPMT*3C) and inosine triphosphate pyrophosphatase (ITPA-C94A), in Caucasian (n = 94, age < 20) and Vietnamese (n = 141, age < 16 years) childhood ALL and (b) to assess the impact of a multilocus genetic risk score (MGRS) on relapse-free survival (RFS) using a Cox proportional-hazards regression model. Results The prevalence of MTHFR-677TT genotype was significantly higher in Caucasians (P = 0.008), in contrast to the prevalence of TYMS-TSER*3R/3R and ITPA-94AA/AC genotypes which were significantly higher in Vietnamese (P < 0.001 and P = 0.02, respectively). Compared with children with a low MGRS (≤3), those with a high MGRS (≥4) were 2.06 (95% CI = 1.01, 4.22; P = 0.04) times more likely to relapse. Adding MGRS into a multivariate Cox regression model with race/ethnicity and four clinical variables improved the predictive accuracy of the model (AUC from 0.682 to 0.709 at 24 months). Conclusion Including MGRS into a clinical model improved the predictive accuracy of short and medium term prognosis, hence confirming the association between well determined pharmacogenotypes and outcome of paediatric ALL. Whether variants on other genes associated with folate metabolism can substantially improve the predictive value of current MGRS is not known but deserves further evaluation. PMID:25099492

A case of recurrent pancytopenia in a patient with acute promyelocytic leukemia on maintenance chemotherapy and concomitant methyltetrahydrofolate reductase and thiopurine S-methyltransferase mutation - review of literature.

PubMed

Keung, Yi-Kong; Keung, Lap-Woon; Hong-Lung Hu, Eddie

2016-06-01

Pharmacogenetics is a study of how genetic variation of an individual affects the drug response. We report a case of recurrent pancytopenia resulting from maintenance chemotherapy in a patient with acute promyelocytic leukemia and two pharmacogenetic mutations, namely, methylene tetrahydrofolate reductase C677T homozygous mutation and thiopurine methyltransferase mutation. © The Author(s) 2015.

Genetics Home Reference: thiopurine S-methyltransferase deficiency

MedlinePlus

... activity: a large, prospective population study. Pharmacogenomics. 2008 Mar;9(3):303-9. doi: 10.2217/14622416. ... genotype and thiopurine dosing. Clin Pharmacol Ther. 2011 Mar;89(3):387-91. doi: 10.1038/clpt. ...

Emerging role of NUDT15 polymorphisms in 6-mercaptopurine metabolism and dose related toxicity in acute lymphoblastic leukaemia.

PubMed

Singh, Minu; Bhatia, Prateek; Khera, Sanjeev; Trehan, Amita

2017-11-01

Despite more than 80% long term survival in ALL, morbidity due to drug related myelotoxicity remains high. Germline variants of thiopurine metabolizing enzymes (TPMT and ITPA) have been described which are associated with increased drug toxicity during maintenance phase, but their prevalence in different ethnic groups is variable to account for relatively high myelotoxicity incidence. NUDT15 variant (rs116855232) has been recently identified as a novel polymorphism related with thiopurine-induced leucopenia in inflammatory bowel disease and ALL. Current review highlights the scientific data on NUDT15 enzyme variant and its relation to 6-MP toxicity in various ethnic populations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Therapeutic drug monitoring of thiopurine metabolites in adult thiopurine tolerant IBD patients on maintenance therapy.

PubMed

Gilissen, Lennard P L; Wong, Dennis R; Engels, Leopold G J B; Bierau, Jörgen; Bakker, Jaap A; Paulussen, Aimée D C; Romberg-Camps, Mariëlle J; Stronkhorst, Arnold; Bus, Paul; Bos, Laurens P; Hooymans, Piet M; Stockbrügger, Reinhold W; Neef, Cees; Masclee, Ad A M

2012-07-01

Therapeutic drug monitoring of active metabolites of thiopurines, azathioprine and 6-mercaptopurine, is relatively new. The proposed therapeutic threshold level of the active 6-thioguanine nucleotides (6-TGN) is ≥235 pmol/8×10(8) erythrocytes. The aim of this prospective cross-sectional study was to compare 6-TGN levels in adult thiopurine tolerant IBD patients with an exacerbation with those in remission, and to determine the therapeutic 6-TGN cut-off level. Hundred IBD patients were included. Outcome measures were thiopurine metabolite levels, calculated therapeutic 6-TGN cut-off level, CDAI/CAI scores, thiopurine dose and TPMT enzyme activity. Forty-one patients had an exacerbation, 59 patients were in remission. In 17% of all patients 6-TGN levels were compatible with non-compliance. The median 6-TGN levels were not significantly different between the exacerbation and remission group (227 versus 263 pmol/8×10(8) erythrocytes, p=0.29). The previous reported therapeutic 6-TGN cut-off level of 235 pmol/8×10(8) erythrocytes was confirmed in this study. Twenty-six of the 41 patients (63%) with active disease had 6-TGN levels below this threshold and 24 of 59 IBD patients (41%) in clinical remission (p=0.04). Thiopurine non-compliance occurs frequently both in active and quiescent disease. 6-TGN levels below or above the therapeutic threshold are associated with a significant higher chance of IBD exacerbation and remission, respectively. These data support the role of therapeutic drug monitoring in thiopurine maintenance therapy in IBD to reveal non-compliance or underdosing, and can be used as a practical tool to optimize thiopurine therapy, especially in case of thiopurine non-response. Copyright © 2011 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Pharmacokinetics of two 6-mercaptopurine liquid formulations in children with acute lymphoblastic leukemia.

PubMed

Tolbert, Jaszianne A; Bai, Shasha; Abdel-Rahman, Susan M; August, Keith J; Weir, Scott J; Kearns, Gregory L; Neville, Kathleen A

2017-08-01

A liquid formulation of 6-mercaptopurine (6-MP) was recently approved by the Food and Drug Administration (Purixan®) based on bioavailability (BA) data from healthy adults. We examined the pharmacokinetics (PK) and BA of 6-MP in children with acute lymphoblastic leukemia (ALL) comparing a marketed tablet, two extemporaneously prepared liquid formulations, and data from the approved liquid formulation. Twenty-two children (6-17 years) participated in a randomized two-way, crossover study of two cohorts. Group 1 (n = 11; five males) received a 5 mg/ml liquid formulation and the marketed 50 mg 6-MP tablet on separate occasions, and Group 2 (n = 11; five males) received a 50 mg/ml liquid formulation and the marketed tablet. The usual prescribed 6-MP dose (25-115 mg/m 2 ) was given after an 8-hr fast. Serial blood samples were collected over 8 hr postdose. Plasma 6-MP concentrations were determined using a good laboratory practice (GLP)-validated liquid chromatography-tandem mass spectrometry method. PK parameters were calculated using noncompartmental analysis and compared within and between cohorts, and thiopurine methyltransferase (TPMT) genotype was analyzed. No patient had a TPMT genotype reflective of a poor metabolizer phenotype. Comparison of PK parameters between 5 and 50 mg/ml treatments revealed significant differences (P <0.05) in AUC N (where AUC is area under the curve), C maxN , and T max . Comparisons within each group revealed significant differences in AUC 0-∞ and T max in the 5 mg/ml group. Pharmacokinetic profiles of 6-MP established in healthy adults with the approved liquid formulation may not reflect the PK profile in children with ALL. Formulation-specific differences in PK may significantly impact the dose-exposure profile in these children and must be considered. © 2017 Wiley Periodicals, Inc.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia.

PubMed

Hawwa, Ahmed F; Collier, Paul S; Millership, Jeff S; McCarthy, Anthony; Dempsey, Sid; Cairns, Carole; McElnay, James C

2008-12-01

To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model. The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype.

Model-Based Individualized Treatment of Chemotherapeutics: Bayesian Population Modeling and Dose Optimization

PubMed Central

Jayachandran, Devaraj; Laínez-Aguirre, José; Rundell, Ann; Vik, Terry; Hannemann, Robert; Reklaitis, Gintaras; Ramkrishna, Doraiswami

2015-01-01

6-Mercaptopurine (6-MP) is one of the key drugs in the treatment of many pediatric cancers, auto immune diseases and inflammatory bowel disease. 6-MP is a prodrug, converted to an active metabolite 6-thioguanine nucleotide (6-TGN) through enzymatic reaction involving thiopurine methyltransferase (TPMT). Pharmacogenomic variation observed in the TPMT enzyme produces a significant variation in drug response among the patient population. Despite 6-MP’s widespread use and observed variation in treatment response, efforts at quantitative optimization of dose regimens for individual patients are limited. In addition, research efforts devoted on pharmacogenomics to predict clinical responses are proving far from ideal. In this work, we present a Bayesian population modeling approach to develop a pharmacological model for 6-MP metabolism in humans. In the face of scarcity of data in clinical settings, a global sensitivity analysis based model reduction approach is used to minimize the parameter space. For accurate estimation of sensitive parameters, robust optimal experimental design based on D-optimality criteria was exploited. With the patient-specific model, a model predictive control algorithm is used to optimize the dose scheduling with the objective of maintaining the 6-TGN concentration within its therapeutic window. More importantly, for the first time, we show how the incorporation of information from different levels of biological chain-of response (i.e. gene expression-enzyme phenotype-drug phenotype) plays a critical role in determining the uncertainty in predicting therapeutic target. The model and the control approach can be utilized in the clinical setting to individualize 6-MP dosing based on the patient’s ability to metabolize the drug instead of the traditional standard-dose-for-all approach. PMID:26226448

Advances and challenges in hereditary cancer pharmacogenetics.

PubMed

Cascorbi, Ingolf; Werk, Anneke Nina

2017-01-01

Cancer pharmacogenetics usually considers tumor-specific targets. However, hereditary genetic variants may interfere with the pharmacokinetics of antimetabolites and other anti-cancer drugs, which may lead to severe adverse events. Areas covered: Here, the impact of hereditary genes considered in drug labels such as thiopurine S-methyltransferase (TPMT), UDP-glucuronosyltransferase 1A1 (UTG1A1) and dihydropyrimidine dehydrogenase (DPYD) are discussed with respect to guidelines of the Clinical Pharmacogenetics Implementation Consortium (CPIC). Moreover, the association between genetic variants of drug transporters with the clinical outcome is comprehensively discussed. Expert opinion: Precision therapy in the field of oncology is developing tremendously. There are a number of somatic tumor genetic markers that are indicative for treatment with anti-cancer drugs. By contrast, for some hereditary variants, recommendations have been developed. Although we have vast knowledge on the association between drug transporter variants and clinical outcome, the overall data is inconsistent and the predictability of the related phenotype is low. Further developments in research may lead to the discovery of rare, but functionally relevant single nucleotide polymorphisms and a better understanding of multiple genomic, epigenomic as well as phenotypic factors, contributing to drug response in malignancies.

[Genetic polymorphisms of thiopurine methyltransferase and incidence of adverse events in patients with medical indication of azathioprine].

PubMed

Buhl, Manuel A; Gómez, Graciela; Collado, María Victoria; Oddo, Elisabet M; Khoury, Marina; Azurmendi, Pablo J; Sarano, Judith

2018-01-01

Azathioprine is a thiopurine which has a narrow therapeutic index and marked hematological and hepatic toxicity. Thiopurine s-methyltransferase is an enzyme involved in the metabolism of thiopurines. Mutations in the gene that encodes the enzyme may augment the risk of adverse events. For that reason, pharmacogenetic determinations prior to the initiation of therapy can provide useful information for the future therapeutic strategy. Nevertheless, its utility in the local environment is not completely established. Forty-five subjects (13 men) who had been prescribed azathioprine were included. The presence of *2, *3A, *3B and *3C mutations were determined by PCR-RFLP, and the relationship between genotype and incidence of adverse events related to the drug was analyzed. Nine carried at least one non-functional allele, one of them with *3A/*3A genotype. Among the eighteen patients who initiated treatment with azathioprine, toxicity was detected in 3 cases: 2 mild events were observed in patients with normal genotype, and the only serious event (bone marrow suppression) occurred in the individual with homozygous mutant genotype. The only homozygous mutant patient developed the most severe of the registered events, in spite of being under treatment with low doses of azathioprine. This is the reason why enzymatic determination could be of utility, even though it does not replace clinical and biochemical follow-up in patients under thiopurine treatment.

The Role of Laboratory Tests in Crohn’s Disease

PubMed Central

Cappello, Maria; Morreale, Gaetano Cristian

2016-01-01

In the past, laboratory tests were considered of limited value in Crohn’s disease (CD). In the era of biologics, laboratory tests have become essential to evaluate the inflammatory burden of the disease (C-reactive protein, fecal calprotectin) since symptoms-based scores are subjective, to predict the response to pharmacological options and the risk of relapse, to discriminate CD from ulcerative colitis, to select candidates to anti-tumor necrosis factors [screening tests looking for hepatitis B virus and hepatitis C virus status and latent tuberculosis], to assess the risk of adverse events (testing for thiopurine metabolites and thiopurine-methyltransferase activity), and to personalize and optimize therapy (therapeutic drug monitoring). Pharmacogenetics, though presently confined to the assessment of thiopurineme methyltransferase polymorphisms and hematological toxicity associated with thiopurine treatment, is a promising field that will contribute to a better understanding of the molecular mechanisms of the variability in response to the drugs used in CD with the attempt to expand personalized care and precision medicine strategies. PMID:27656094

Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry.

PubMed

Moriyama, Takaya; Yang, Yung-Li; Nishii, Rina; Ariffin, Hany; Liu, Chengcheng; Lin, Ting-Nien; Yang, Wenjian; Lin, Dong-Tsamn; Yu, Chih-Hsiang; Kham, Shirley; Pui, Ching-Hon; Evans, William E; Jeha, Sima; Relling, Mary V; Yeoh, Allen Eng-Juh; Yang, Jun J

2017-09-07

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT ). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Children's Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations. © 2017 by The American Society of Hematology.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine.

PubMed

Hawwa, Ahmed F; Millership, Jeff S; Collier, Paul S; Vandenbroeck, Koen; McCarthy, Anthony; Dempsey, Sid; Cairns, Carole; Collins, John; Rodgers, Colin; McElnay, James C

2008-10-01

To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94-->A and IVS2+21A-->C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A-->C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A-->C variants with thrombocytopenia (P = 0.012). CONCLUSIONS; Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.

Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations of the thiopurine S-methyltransferase reaction with 6-mercaptopurine.

PubMed

Pan, Xiao-Liang; Cui, Feng-Chao; Liu, Jing-Yao

2011-06-23

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/mol, which is in good agreement with the available experimental rate constant data.

New genetic associations in thiopurine-related bone marrow toxicity among inflammatory bowel disease patients.

PubMed

Zabala, William; Cruz, Raquel; Barreiro-de Acosta, Manuel; Chaparro, María; Panes, Julián; Echarri, Ana; Esteve, Maria; Carpio, Daniel; Andreu, Montserrat; García-Planella, Esther; Domenech, Eugeni; Carracedo, Angel; Gisbert, Javier P; Barros, Francisco

2013-04-01

The toxicity related to thiopurine drug therapy for inflammatory bowel disease (IBD) varies widely among patients. Almost 15-30% of patients with IBD develop side effects during treatment, often bone marrow suppression. Several factors have been implicated in determining this toxicity, mainly individual genetic variation related to formation of active thiopurine metabolites. The aim was to identify genes involved in thiopurine-related myelosuppression. A two-stage investigation of 19,217 coding SNPs (cSNPs) was performed in a Spanish (Inflammatory Bowel Disease Group of Galicia [EIGA]) cohort of 173 IBD patients, 15 with bone marrow suppression. The top 20 cSNPs identified in the first stage with p < 10(-3) for allelic test association and SNPs that define the common TPMT alleles were replicated in a different Spanish (ENEIDA) cohort (87 patients, 29 with bone marrow suppression). Several cSNPs showed a significant p-value in the allelic joint analysis (p-Cochran-Mantel-Haenszel test ≤2.55 × 10(-3)) despite no cSNP passing correction for multiple testing in the first cohort. Of note is rs3729961 in the gene IL6ST, a transducer signal chain shared by many cytokines including IL6 (p-value combined = 2.36 × 10(-4), odds ratio [95% CI]: 3.41 [1.71-6.78]). In addition, we detected association with rs3749598 in the FSTL5 gene that appears to interact with metalloproteases at the extracellular matrix level (p-value combined = 4.89 × 10(-4)), odds ratio (95% CI): 3.67 (1.68-8.01). We have identified IL6ST and FSLT5 as new bone marrow suppression susceptibility candidate genes after thiopurine treatment in IBD patients. This is the first report of variants associated with thiopurine-related myelosuppression that was identified by a genome-wide association study. Its validation awaits functional analyses and replication in additional studies. Original submitted 14 September 2012; Revision submitted 13 February 2013.

Development and use of active clinical decision support for preemptive pharmacogenomics

PubMed Central

Bell, Gillian C; Crews, Kristine R; Wilkinson, Mark R; Haidar, Cyrine E; Hicks, J Kevin; Baker, Donald K; Kornegay, Nancy M; Yang, Wenjian; Cross, Shane J; Howard, Scott C; Freimuth, Robert R; Evans, William E; Broeckel, Ulrich; Relling, Mary V; Hoffman, James M

2014-01-01

Background Active clinical decision support (CDS) delivered through an electronic health record (EHR) facilitates gene-based drug prescribing and other applications of genomics to patient care. Objective We describe the development, implementation, and evaluation of active CDS for multiple pharmacogenetic test results reported preemptively. Materials and methods Clinical pharmacogenetic test results accompanied by clinical interpretations are placed into the patient's EHR, typically before a relevant drug is prescribed. Problem list entries created for high-risk phenotypes provide an unambiguous trigger for delivery of post-test alerts to clinicians when high-risk drugs are prescribed. In addition, pre-test alerts are issued if a very-high risk medication is prescribed (eg, a thiopurine), prior to the appropriate pharmacogenetic test result being entered into the EHR. Our CDS can be readily modified to incorporate new genes or high-risk drugs as they emerge. Results Through November 2012, 35 customized pharmacogenetic rules have been implemented, including rules for TPMT with azathioprine, thioguanine, and mercaptopurine, and for CYP2D6 with codeine, tramadol, amitriptyline, fluoxetine, and paroxetine. Between May 2011 and November 2012, the pre-test alerts were electronically issued 1106 times (76 for thiopurines and 1030 for drugs metabolized by CYP2D6), and the post-test alerts were issued 1552 times (1521 for TPMT and 31 for CYP2D6). Analysis of alert outcomes revealed that the interruptive CDS appropriately guided prescribing in 95% of patients for whom they were issued. Conclusions Our experience illustrates the feasibility of developing computational systems that provide clinicians with actionable alerts for gene-based drug prescribing at the point of care. PMID:23978487

The Relationship of Genetics, Nursing Practice, and Informatics Tools in 6-Mercaptopurine Dosing in Pediatric Oncology [Formula: see text].

PubMed

Haylett, Wendy J

An antileukemic agent prescribed for pediatric oncology patients during the maintenance phase of therapy for acute lymphoblastic leukemia, 6-mercaptopurine (6-MP), is highly influenced by genetic variations in the thiopurine S-methyltransferase enzyme. As such, 6-MP must be dosed so that patients with 1 or 2 inactive thiopurine S-methyltransferase alleles will not incur an increased risk for myelosuppression or other toxicities. Informatics tools such as clinical decision support systems are useful for the application of this and similar pharmacogenetics information to the realm of nursing and clinical practice for safe and effective patient care. This article will discuss pharmacogenetics and the associated use of 6-MP; present implications for nursing practice; identify informatics tools such as clinical decision support systems, which can greatly enhance the care of patients whose treatment is based on critical genetic information; and examine the relationship of genetics, nursing practice, and informatics for 6-MP dosing in pediatric oncology.

State-of-the-art: Immunosuppression and biologic therapy.

PubMed

Sandborn, William J

2010-01-01

Azathioprine and 6-mercaptopurine are orally administered immunosuppressive drugs which are effective for the treatment of Crohn's disease and ulcerative colitis. Azathioprine is rapidly converted to 6-mercaptopurine after administration. 6-Mercaptopurine is then either converted to the putative active metabolites, the 6-thioguinine nucleotides, or inactivated by the enzyme xanthine oxidase to 6-thiouric acid or alternatively inactivated to 6-methylmercaptopurine by the enzyme thiopurine methyltransferase. Thiopurine methyltransferase activity is genetically determined, with one in 300 patients having low or absent enzyme activity, one in 10 patients having intermediate enzyme activity, and 9 in 10 patients having normal enzyme activity. Patients with intermediate or low thiopurine methyltransferase activity are at risk for early leukopenia. Higher erythrocyte 6-thioguinine nucleotide concentrations are associated with a greater likelihood of clinical response. Azathioprine is modestly effective for Crohn's disease and ulcerative colitis. Toxicity associated with azathioprine includes infection and lymphoma. Anti-TNF therapy with infliximab, adalimumab, and certolizumab pegol is effective for induction and maintenance treatment of Crohn's disease, and infliximab is effective for ulcerative colitis. Toxicity associated with anti-TNF therapy includes infection and lymphoma. Combination therapy with infliximab and azathioprine is more effective for inducing and maintaining steroid-free remission and mucosal healing then monotherapy with either drug alone. Strategies to reduce immunogenicity of anti-TNF agents include combination therapy with azathioprine and administration of a loading dose followed by systematic maintenance dosing. Higher serum trough concentrations of infliximab occur more frequently in patients receiving combination therapy with azathioprine and are associated with better clinical outcomes. Combination therapy is associated with an increased relative risk of opportunistic infection, but is not associated with an increased absolute risk of serious infection. Clinical practice should change such that combination therapy with an anti-TNF agent and azathioprine replace azathioprine in patients failing first line therapy with mesalamine and/or steroids. Copyright © 2010 S. Karger AG, Basel.

PACSIN2 polymorphism is associated with thiopurine-induced hematological toxicity in children with acute lymphoblastic leukaemia undergoing maintenance therapy.

PubMed

Smid, Alenka; Karas-Kuzelicki, Natasa; Jazbec, Janez; Mlinaric-Rascan, Irena

2016-07-25

Adequate maintenance therapy for childhood acute lymphoblastic leukemia (ALL), with 6-mercaptopurine as an essential component, is necessary for retaining durable remission. Interruptions or discontinuations of the therapy due to drug-related toxicities, which can be life threatening, may result in an increased risk of relapse. In this retrospective study including 305 paediatric ALL patients undergoing maintenance therapy, we systematically investigated the individual and combined effects of genetic variants of folate pathway enzymes, as well as of polymorphisms in PACSIN2 and ITPA, on drug-induced toxicities by applying a multi-analytical approach including logistic regression (LR), classification and regression tree (CART) and generalized multifactor dimensionality reduction (GMDR). In addition to the TPMT genotype, confirmed to be a major determinant of drug related toxicities, we identified the PACSIN2 rs2413739TT genotype as being a significant risk factor for 6-MP-induced toxicity in wild-type TPMT patients. A gene-gene interaction between MTRR (rs1801394) and MTHFR (rs1801133) was detected by GMDR and proved to have an independent effect on the risk of stomatitis, as shown by LR analysis. To our knowledge, this is the first study showing PACSIN2 genotype association with hematological toxicity in ALL patients undergoing maintenance therapy.

Assessment of mercaptopurine (6MP) metabolites and 6MP metabolic key-enzymes in childhood acute lymphoblastic leukemia.

PubMed

Wojtuszkiewicz, Anna; Barcelos, Ana; Dubbelman, Boas; De Abreu, Ronney; Brouwer, Connie; Bökkerink, Jos P; de Haas, Valerie; de Groot-Kruseman, Hester; Jansen, Gerrit; Kaspers, Gertjan L; Cloos, Jacqueline; Peters, G J

2014-01-01

Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MP's sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N=236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5'-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP--TGN and meTIN--showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions.

NUDT15 p.R139C variant is common and strongly associated with azathioprine-induced early leukopenia and severe alopecia in Korean patients with various neurological diseases.

PubMed

Kim, Sun-Young; Shin, Jin-Hong; Park, Jin-Sung; Kang, Sa-Yoon; Nam, Tai-Seung; Kim, Jong Kuk; Park, Ki-Jong; Huh, So-Young; Oh, Ji Seon; Kang, Boram; Kim, Dae-Seong

2017-07-15

Azathioprine (AZA)-induced leukopenia is a relatively common complication in Korean patients. In addition to variation in TPMT (thiopurine S-methyltransferase), the NUDT15 p.R139C variant was recently identified to have a strong association with AZA-induced leukopenia. We investigated these associations in Korean patients undergoing AZA treatment with various neurological diseases. Among 84 enrolled patients, 20 (23.8%; 7 early, 13 late) exhibited leukopenia. The NUDT15 p.R139C variant was associated with leukopenia (OR: 11.844, 95% CI 3.984-36.024, p=1.327 × 10 -5 ). The allelic frequency of NUDT15 p.R139C was as high as 10.7% and the frequency of the C/C, C/T, and T/T genotypes was 84.5, 10.7, and 5.9%, respectively. All T/T homozygous patients (5/5) developed early severe-grade leukopenia (white blood cells <1000mm -3 ) and severe alopecia. NUDT15 p.R139C was strongly associated with early leukopenia and severe alopecia (OR for early leukopenia: 107.624, 95% CI 18.857-614.250, p=1.403 × 10 -7 , OR for severe alopecia: 77.152, 95% CI 17.378-342.526, p=1.101 × 10 -8 ). The sensitivity and specificity for predicting AZA-induced early leukopenia were 85.7% and 92.2%, respectively. Therefore, the NUDT15 p.R139C variant is common and strongly associated with AZA-induced early leukopenia and severe alopecia in Korean patients with various neurological diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Early prediction of thiopurine-induced hepatotoxicity in inflammatory bowel disease.

PubMed

Wong, D R; Coenen, M J H; Derijks, L J J; Vermeulen, S H; van Marrewijk, C J; Klungel, O H; Scheffer, H; Franke, B; Guchelaar, H-J; de Jong, D J; Engels, L G J B; Verbeek, A L M; Hooymans, P M

2017-02-01

Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations. To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment. The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5. Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 10 8 erythrocytes was defined. Analysis of patients on stable thiopurine dose (n = 174) showed that those exceeding the 6-MMPR threshold were at increased risk of hepatotoxicity: OR = 3.8 (95% CI: 1.8-8.0). Age, male gender and BMI were significant determinants. A predictive algorithm was developed based on these determinants and the 6-MMPR threshold to assess hepatotoxicity risk [AUC = 0.83 (95% CI: 0.75-0.91)]. 6-MMPR concentrations above the threshold also correlated with gastrointestinal complaints: OR = 2.4 (95% CI: 1.4-4.3), and general malaise: OR = 2.0 (95% CI: 1.1-3.7). In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure. © 2016 John Wiley & Sons Ltd.

SLCO1B1 Polymorphisms are Associated With Drug Intolerance in Childhood Leukemia Maintenance Therapy.

PubMed

Eldem, İrem; Yavuz, Duygu; Cumaoğullari, Özge; İleri, Talia; Ünal İnce, Elif; Ertem, Mehmet; Doğanay Erdoğan, Beyza; Bindak, Recep; Özdağ, Hilal; Şatiroğlu-Tufan, N Lale; Uysal, L Zümrüt

2018-04-20

Therapy discontinuations and toxicities occur because of significant interindividual variations in 6-mercaptopurine (6-MP) and methotrexate (MTX) response during maintenance therapy of childhood acute lymphoblastic leukemia (ALL). 6-MP/MTX intolerance in some of the patients cannot be explained by thiopurine S-methyl transferase (TPMT) gene variants. In this study, we aimed to investigate candidate pharmacogenetic determinants of 6-MP and MTX intolerance in Turkish ALL children. In total, 48 children with ALL who had completed or were receiving maintenance therapy according to Children's Oncology Group (COG) protocols were enrolled. Fifteen single-nucleotide polymorphisms in 8 candidate genes that were related to drug toxicity or had a role in the 6-MP/MTX metabolism (TPMT, ITPA, MTHFR, IMPDH2, PACSIN2, SLCO1B1, ABCC4, and PYGL) were genotyped by competitive allele-specific PCR (KASP). Drug doses during maintenance therapy were modified according to the protocol. The median drug dose intensity was 50% (28% to 92%) for 6-MP and 58% (27% to 99%) for MTX in the first year of maintenance therapy, which were lower than that scheduled in all patients. Among the analyzed polymorphisms, variant alleles in SLCO1B1 rs4149056 and rs11045879 were found to be associated with lower 6-MP/MTX tolerance. SLCO1B1 rs4149056 and rs11045879 polymorphisms may be important genetic markers to individualize 6-MP/MTX doses.

Combined Detection of NUDT15 Variants Could Highly Predict Thiopurine-induced Leukopenia in Chinese Patients with Inflammatory Bowel Disease: A Multicenter Analysis.

PubMed

Chao, Kang; Wang, Xueding; Cao, Qian; Qian, Jiaming; Wu, Kaichun; Zhu, Xia; Yang, Hong; Liang, Jie; Lin, Lang; Huang, Zicheng; Zhang, Yu; Huang, Yibiao; Sun, Yinghao; Xue, Xianmin; Huang, Min; Hu, Pinjin; Lan, Ping; Gao, Xiang

2017-09-01

NUDT15 c.415C>T was a novel genetic marker confirmed in our center for thiopurine-induced leukopenia in Chinese inflammatory bowel disease (IBD). For validation, a large cohort study is needed. Meanwhile, the newly discovered NUDT15 coding variants (c.36_37insGGAGTC and c.52 G>A) have not been studied in patients with IBD. We aimed to further confirm the influence of 3 NUDT15 variants (c.415C>T, c.36_37insGGAGTC, and c.52G>A) on thiopurine-induced leukopenia in Chinese patients with IBD. Patients prescribed on thiopurines for at least 2 weeks were recruited from 4 tertiary hospitals. Clinical data were collected. NUDT15 genotypes were determined with polymerase chain reaction-RFLP and sequencing. The interactions between variants and leukopenia were analyzed. A total of 732 patients were included, 177 (24.3%) of whom developed leukopenia. There were strong associations of NUDT15 c.415C>T, c.36_37insGGAGTC, and c.52G>A with thiopurine-induced leukopenia (P = 1.81 × 10, P = 4.74 × 10 and P = 0.04, respectively), whereas there was no relevance for thiopurine S-methyltransferase genotypes (P = 0.25). The predictive sensitivity of NUDT15 c.415C>T was 49.2%, whereas it increased to 55.4% when combined analysis with c.36_37insGGAGTC and c.52G>A. Notably, not only the homozygotes with NUDT15 c.415C>T but also the heterozygotes both carrying c.415C>T and c.52G>A developed early leukopenia. The median dosage for NUDT15 c.415C>T carriers was significantly lower than that for wild-type (P < 0.001). We confirmed that NUDT15 c.415C>T, c.36_37insGGAGTC, and c.52G>A variants were risk factors for thiopurine-induced leukopenia. Combined detection of the 3 variants could increase the predictive sensitivity of thiopurine-induced leukopenia and help to distinguish early leukopenia in heterozygote of c.415C>T in Chinese patients with IBD. Treatment monitoring by NUDT15 variants may be promising in individualized therapy.

Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine.

PubMed

Zhang, Wei; Modén, Olof; Mannervik, Bengt

2010-07-30

Azathioprine has been clinically used for decades in connection with organ transplantation, autoimmune disease, and treatment of cancer. Toxic side-reactions are common and have been linked to the liberation of excessively high concentrations of 6-mercaptopurine and corresponding toxic metabolites. An allelic variant of thiopurine methyltransferase with low activity is associated with elevated concentrations of 6-mercaptopurine. However, other genetic markers remain to be identified in order to fully account for adverse reactions and efficacy failure. In the present study, we studied the five known allelic variants of human glutathione transferase A2-2 (GST A2-2) (EC 2.5.1.18), abundantly expressed in liver and efficiently catalyzing the bioactivation of azathioprine to release 6-mercaptopurine. All five variants exhibited high activity with azathioprine, but allelic variant E of GST A2-2 displayed a 3-4-fold elevated catalytic efficiency compared to the other variants. High GST activity can lead to overproduction of 6-mercaptopurine, and the nature of the multiple forms of GSTs in a patient will obviously affect the metabolism of azathioprine. In addition to GST A2-2, the polymorphic GST M1-1 is also highly active with azathioprine. Considering our findings, it appears that the genotypic and phenotypic variations in the GST complement may influence the presentation of adverse reactions in patients treated with azathioprine. Clinical trials will be required to clarify the impact of the GST expression in comparison with the established biomarker thiopurine methyltransferase as predictors of adverse reactions. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner.

PubMed

Kotur, Nikola; Stankovic, Biljana; Kassela, Katerina; Georgitsi, Marianthi; Vicha, Anna; Leontari, Iliana; Dokmanovic, Lidija; Janic, Dragana; Krstovski, Nada; Klaassen, Kristel; Radmilovic, Milena; Stojiljkovic, Maja; Nikcevic, Gordana; Simeonidis, Argiris; Sivolapenko, Gregory; Pavlovic, Sonja; Patrinos, George P; Zukic, Branka

2012-02-01

TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Pharmacogenetic Predictors of Treatment-Related Toxicity Among Children With Acute Lymphoblastic Leukemia.

PubMed

Maxwell, Rochelle R; Cole, Peter D

2017-06-01

The aim of this review is to summarize the most recent and most robust pharmacogenetic predictors of treatment-related toxicity (TRT) in childhood acute lymphoblastic leukemia (ALL). Multiple studies have examined the toxicities of the primary chemotherapeutic agents used to treat childhood ALL in relation to host genetic factors. However, few results have been replicated independently, largely due to cohort differences in ancestry, chemotherapy treatment protocols, and definitions of toxicities. To date, there is only one widely accepted clinical guideline for dose modification based on gene status: thiopurine dosing based on TPMT genotype. Based on recent data, it is likely that this guideline will be modified to incorporate other gene variants, such as NUDT15. We highlight genetic variants that have been consistently associated with TRT across treatment groups, as well as those that best illustrate the underlying pathophysiology of TRT. In the coming decade, we expect that survivorship care will routinely specify screening recommendations based on genetics. Furthermore, clinical trials testing protective interventions may modify inclusion criteria based on genetically determined risk of specific TRTs.

[Acute lymphoblastic leukemia: a genomic perspective].

PubMed

Jiménez-Morales, Silvia; Hidalgo-Miranda, Alfredo; Ramírez-Bello, Julián

In parallel to the human genome sequencing project, several technological platforms have been developed that let us gain insight into the genome structure of human entities, as well as evaluate their usefulness in the clinical approach of the patient. Thus, in acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, genomic tools promise to be useful to detect patients at high risk of relapse, either at diagnosis or during treatment (minimal residual disease), and they also increase the possibility to identify cases at risk of adverse reactions to chemotherapy. Therefore, the physician could offer patient-tailored therapeutic schemes. A clear example of the useful genomic tools is the identification of single nucleotide polymorphisms (SNPs) in the thiopurine methyl transferase (TPMT) gene, where the presence of two null alleles (homozygous or compound heterozygous) indicates the need to reduce the dose of mercaptopurine by up to 90% to avoid toxic effects which could lead to the death of the patient. In this review, we provide an overview of the genomic perspective of ALL, describing some strategies that contribute to the identification of biomarkers with potential clinical application. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Update 2014: advances to optimize 6-mercaptopurine and azathioprine to reduce toxicity and improve efficacy in the management of IBD.

PubMed

Amin, Jaimin; Huang, Brian; Yoon, Jessica; Shih, David Q

2015-02-01

The thiopurine drugs, 6-mercaptopurine (6-MP) and azathioprine (AZA), remain as a mainstay therapy in inflammatory bowel disease (IBD). Differences in metabolism of these drugs lead to individual variation in thiopurine metabolite levels that can determine its therapeutic efficacy and development of adverse reactions. In this update, we will review thiopurine metabolic pathway along with the up-to-date approaches in administering thiopurine medications based on the current literature. A search of the PubMed database by 2 independent reviewers identifying 98 articles evaluating thiopurine metabolism and IBD management. Monitoring thiopurine metabolites can assist physicians in optimizing 6-MP and AZA therapy in treating patients with IBD. Of the dosing strategies reviewed, we found evidence for monitoring thiopurine metabolite level, use of allopurinol with thiopurine, use of mesalamine with thiopurine, combination therapy with thiopurine and anti-tumor necrosis factor agents, and split dosing of AZA or 6-MP to optimize thiopurine therapy and minimize adverse effects in IBD. Based on the currently available literature, various dosing strategies to improve therapeutic response and reduce adverse reactions can be considered, including use of allopurinol with thiopurine, use of mesalamine with thiopurine, combination therapy with thiopurine and anti-tumor necrosis factor agents, and split dosing of thiopurine.

Relevance of pharmacogenetic aspects of mercaptopurine metabolism in the treatment of interstitial lung disease.

PubMed

Bakker, Jaap A; Drent, Marjolein; Bierau, Jörgen

2007-09-01

Mercaptopurine therapy is increasingly important as immunosuppressive therapy in interstitial lung disease. We focus on human mercaptopurine metabolism and the defects in this metabolism causing adverse drug reactions. Defects in mercaptopurine metabolizing enzymes like thiopurine methyltransferase and inosine triphosphate pyrophosphohydrolase lead to severe adverse drug reactions, sometimes with fatal outcome. Other enzymes, still not thoroughly investigated, can give rise to toxic effects or decreased efficacy in mercaptopurine therapy when the activity of these enzymes is altered. Pharmacogenetic screening of potential patients for mercaptopurine therapy is important to avoid adverse drug reactions caused by inherited enzyme deficiencies in these metabolic pathways. Pretreatment screening for deficiencies of mercaptopurine metabolizing enzymes will significantly reduce the number of patients with an adverse drug reaction and concomitantly associated healthcare costs.

Thiopurines and inflammatory bowel disease: Current evidence and a historical perspective.

PubMed

Axelrad, Jordan E; Roy, Abhik; Lawlor, Garrett; Korelitz, Burton; Lichtiger, Simon

2016-12-14

The use of thiopurines in inflammatory bowel disease (IBD) has been examined in numerous prospective, controlled trials, with a majority demonstrating a clinical benefit. We conducted this review to describe the historical and current evidence in the use of thiopurines in IBD. A systematic search was performed on MEDLINE between 1965 and 2016 to identify studies on thiopurines in IBD. The most robust evidence for thiopurines in IBD includes induction of remission in combination with anti-tumor necrosis factor (anti-TNF) agents, and maintenance of remission and post-operative maintenance in Crohn's disease. Less evidence exists for thiopurine monotherapy in induction of remission, maintenance of ulcerative colitis, chemoprevention of colorectal cancer, and in preventing immunogenicity to anti-TNF. Evidence was often limited by trial design. Overall, thiopurines have demonstrated efficacy in a broad range of presentations of IBD. With more efficacious novel therapeutic agents, the positioning of thiopurines in the management of IBD will change and future studies will analyze the benefit of thiopurines alone and in conjunction with these new medications.

Biomarkers of adverse drug reactions.

PubMed

Carr, Daniel F; Pirmohamed, Munir

2018-02-01

Adverse drug reactions can be caused by a wide range of therapeutics. Adverse drug reactions affect many bodily organ systems and vary widely in severity. Milder adverse drug reactions often resolve quickly following withdrawal of the casual drug or sometimes after dose reduction. Some adverse drug reactions are severe and lead to significant organ/tissue injury which can be fatal. Adverse drug reactions also represent a financial burden to both healthcare providers and the pharmaceutical industry. Thus, a number of stakeholders would benefit from development of new, robust biomarkers for the prediction, diagnosis, and prognostication of adverse drug reactions. There has been significant recent progress in identifying predictive genomic biomarkers with the potential to be used in clinical settings to reduce the burden of adverse drug reactions. These have included biomarkers that can be used to alter drug dose (for example, Thiopurine methyltransferase (TPMT) and azathioprine dose) and drug choice. The latter have in particular included human leukocyte antigen (HLA) biomarkers which identify susceptibility to immune-mediated injuries to major organs such as skin, liver, and bone marrow from a variety of drugs. This review covers both the current state of the art with regard to genomic adverse drug reaction biomarkers. We also review circulating biomarkers that have the potential to be used for both diagnosis and prognosis, and have the added advantage of providing mechanistic information. In the future, we will not be relying on single biomarkers (genomic/non-genomic), but on multiple biomarker panels, integrated through the application of different omics technologies, which will provide information on predisposition, early diagnosis, prognosis, and mechanisms. Impact statement • Genetic and circulating biomarkers present significant opportunities to personalize patient therapy to minimize the risk of adverse drug reactions. ADRs are a significant heath issue and represent a significant burden to patients, healthcare providers, and the pharmaceutical industry. • This review details the current state of the art in biomarkers of ADRs (both genetic and circulating). There is still significant variability in patient response which cannot be explained by current knowledge of genetic risk factors for ADRs; however, we discussed how specific advances in genomics have the potential to yield better and more predictive models. • Many current clinically utilized circulating biomarkers of tissue injury are valid biomarkers for a number of ADRs. However, they often give little insight into the specific cell or tissue subtype which may be affected. Emerging circulating biomarkers with potential to provide greater information on the etiology/pathophysiology of ADRs are described.

Predictors of Thiopurine Treatment Failure in Biologic-Naïve Ulcerative Colitis Patients.

PubMed

Thapa, Sudeep Dhoj; Hadid, Hiba; Usman, Mohammed; Imam, Waseem; Hassan, Ahmad; Schairer, Jason; Jafri, Syed-Mohammed R; Kaur, Nirmal

2016-01-01

Thiopurines (azathioprine and 6-mercaptopurine) have been used in the management of UC patients for over three decades. Nearly half of patients with UC treated with thiopurines fail to achieve remission or lose remission during treatment. Factors associated with thiopurine failure are poorly understood. The primary aim of our study was to investigate patient-related factors which are associated with thiopurine failure. TNF-alpha antagonist-naïve patients with histological diagnosis of UC, receiving thiopurine therapy, with follow-up data from 1 to 3 years were included in the study. Data regarding demographics, laboratory results, and disease characteristics were collected. The primary endpoint was failure of thiopurine therapy, defined as treatment with steroids, therapeutic escalation to TNF-alpha antagonist therapy, or need for surgery. Of the 563 patients identified using ICD-9 codes, 78 TNF-alpha antagonist-naïve patients with a histological diagnosis of UC, receiving thiopurine treatment, were identified. Over the three-year follow-up period, 38 patients failed thiopurine treatment. On adjusted Cox regression, BMI < 25 kg/m(2) (HR 3, 95 % CI 1.55-5.83; p value = 0.001) was significantly associated with thiopurine failure. Furthermore, although not statistically significant, there was a strong trend toward thiopurine failure among patients with serum albumin level < 4 g/dL (HR 1.98, 95 % CI 0.97-4; p value = 0.06), non-smoking status (HR 2.2, 95 % CI 0.96-5.06; p value = 0.06), and higher degree of colon inflammation (HR 1.49, 95 % CI 0.96-2.32; p value = 0.08). Our results show that low body mass index is associated with increased risk of failure of thiopurine treatment. Furthermore, there was a strong trend toward thiopurine failure among patients with low serum albumin level (<4gm/dL). These factors should be considered as markers of non-response to thiopurine monotherapy for patients with moderately severe ulcerative colitis.

Low-dose thiopurine with allopurinol co-therapy overcomes thiopurine intolerance and allows thiopurine continuation in inflammatory bowel disease.

PubMed

Vasudevan, Abhinav; Beswick, Lauren; Friedman, Antony B; Moltzen, Alicia; Haridy, James; Raghunath, Ajay; Sparrow, Miles; van Langenberg, Daniel

2018-02-10

To assess the utility and tolerability of thiopurine-allopurinol co-therapy in inflammatory bowel disease (IBD) patients with intolerance to thiopurine monotherapy. A retrospective observational study assessed cases of thiopurine intolerance then switched to thiopurine allopurinol co-therapy between 2011 and 2015 at two centres. Indications for switch, dosing and subsequent clinical outcomes (including thiopurine persistence) were recorded. Of 767 patients on thiopurines for IBD, 89 (12%) were switched to co-therapy for intolerance. 64/89 (72%) had Crohn's disease, 38 (43%) were males, median age at switch was 40y (range 17-78), median IBD duration 6y (0-29). Median follow-up was 1.9y (0-5). Reasons for switching to co-therapy included fatigue (37%), hepatotoxicity (23%), nausea (23%), arthralgia (10%), headache (12%) and hypersensitivity reaction (4%). Overall, 66 (74%) patients remained on co-therapy until most recent review and achieved a clinical response. High rates of overcoming intolerance (62-100%) occurred with co-therapy for all reasons above, although fatigue was less amenable to switching than non-fatigue indications (62% vs 91%, p = <0.001). Of 34 patients not escalated to biologics with endoscopic data, 15 were in remission (44%) at most recent review. Low-dose thiopurine combined with allopurinol appears safe and effective in overcoming intolerances to thiopurine monotherapy in many cases. Copyright © 2018. Published by Elsevier Ltd.

Thiopurines and inflammatory bowel disease: Current evidence and a historical perspective

PubMed Central

Axelrad, Jordan E; Roy, Abhik; Lawlor, Garrett; Korelitz, Burton; Lichtiger, Simon

2016-01-01

The use of thiopurines in inflammatory bowel disease (IBD) has been examined in numerous prospective, controlled trials, with a majority demonstrating a clinical benefit. We conducted this review to describe the historical and current evidence in the use of thiopurines in IBD. A systematic search was performed on MEDLINE between 1965 and 2016 to identify studies on thiopurines in IBD. The most robust evidence for thiopurines in IBD includes induction of remission in combination with anti-tumor necrosis factor (anti-TNF) agents, and maintenance of remission and post-operative maintenance in Crohn’s disease. Less evidence exists for thiopurine monotherapy in induction of remission, maintenance of ulcerative colitis, chemoprevention of colorectal cancer, and in preventing immunogenicity to anti-TNF. Evidence was often limited by trial design. Overall, thiopurines have demonstrated efficacy in a broad range of presentations of IBD. With more efficacious novel therapeutic agents, the positioning of thiopurines in the management of IBD will change and future studies will analyze the benefit of thiopurines alone and in conjunction with these new medications. PMID:28028358

Thiopurine use associated with reduced B and natural killer cells in inflammatory bowel disease

PubMed Central

Lord, James D; Shows, Donna M

2017-01-01

AIM To identify which blood and mucosal lymphocyte populations are specifically depleted by thiopurine use in vivo. METHODS The thiopurines azathioprine and 6-mercaptopurine have been a mainstay of inflammatory bowel disease (IBD) therapy for decades, but their mechanism of action in vivo remains obscure. Although thiopurines are lymphotoxic at high doses, and have been reported to cause T cell apoptosis in vitro, their ability to control IBD at lower doses suggests that they may selectively deplete particular lymphocyte populations. Blood cells from 19 IBD patients on a thiopurine, 19 IBD patients not on a thiopurine, and 38 matched healthy control subjects were analyzed by multiple multi-color flow cytometry panels to quantify the immune cell subsets contained therein, both as a percent of cells, and as an absolute cell count. Similar analyses were performed on colon biopsies from 17 IBD patients on a thiopurine, 17 IBD patients not on a thiopurine, and 49 healthy screening colonoscopy recipients. RESULTS Complete blood counts revealed lower lymphocyte, but not monocyte or granulocyte, counts in IBD patients who were taking thiopurines at the time of sampling. This reduction was restricted to CD3-negative lymphocytes, wherein both natural killer (NK) and B cells were significantly reduced among thiopurine recipients. Among CD19+ B cells, the transitional B cells were particularly depleted, being nearly absent in both blood and colon biopsies of thiopurine recipients. No differences were associated with thiopurine use in CD8+ T cells, mucosa-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, gamma/delta T cells, Th1, Th17, regulatory T cells (Tregs) or naïve CD4+ T cells. However, patients with IBD had significantly more circulating FOXP3+, Helios+ Tregs and fewer iNKT and MAIT cells than healthy controls. CONCLUSION Thiopurine use is associated with reduced B and NK cell, but not T cell, subpopulations in the blood of IBD patients. PMID:28566883

Excess risk of urinary tract cancers in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study.

PubMed

Bourrier, A; Carrat, F; Colombel, J-F; Bouvier, A-M; Abitbol, V; Marteau, P; Cosnes, J; Simon, T; Peyrin-Biroulet, L; Beaugerie, L

2016-01-01

The risk of urinary tract cancers, including kidney and bladder cancers, was increased in transplant recipients receiving thiopurines. To assess the risk of urinary tract cancers in patients with inflammatory bowel disease (IBD) receiving thiopurines in the CESAME observational cohort. Between May 2004 and June 2005, 19 486 patients with IBD, 30.1% of whom were receiving thiopurines, were enrolled. Median follow-up was 35 months (IQR: 29-40). Ten and six patients developed respectively kidney and bladder cancer. The incidence rates of urinary tract cancer were 0.48/1000 patient-years in patients receiving thiopurines (95% CI: 0.21-0.95), 0.10/1000 patient-years in patients who discontinued thiopurines (95% CI: 0.00-0.56) and 0.30/1000 patient-years in patients never treated with thiopurines (95% CI: 0.12-0.62) at entry. The standardised incidence ratio of urinary tract cancer was 3.40 (95% CI: 1.47-6.71, P = 0.006) in patients receiving thiopurines, 0.64 (95% CI: 0.01-3.56, P = 0.92) in patients previously exposed to thiopurines and 1.17 (95% CI: 0.47-12.42, P = 0.78) in patients never treated with thiopurines. The multivariate-adjusted hazard ratio (HR) of urinary tract cancer between patients receiving thiopurines and those not receiving thiopurines was 2.82 (95% CI: 1.04-7.68, P = 0.04). Other significant risk factors were male gender (HR: 3.98, 95% CI: 1.12-14.10, P = 0.03) and increasing age (HR after 65 years (ref <50): 13.26, 95% CI: 3.52-50.03, P = 0.0001). Patients with IBD receiving thiopurines have an increased risk of urinary tract cancers. Clinically relevant excess risk is observed in older men. © 2015 John Wiley & Sons Ltd.

The role of thiopurine metabolites in inflammatory bowel disease and rheumatological disorders.

PubMed

Friedman, Antony B; Sparrow, Miles P; Gibson, Peter R

2014-02-01

Thiopurines have been a cornerstone of medical management of patients with inflammatory bowel disease(IBD) and many rheumatological disorders. The thiopurines are metabolized to their end products, 6-methymercaptopurine (6MMP) and the 6-thioguanine nucleotides (6TGN), with 6TGN being responsible for thiopurine efficacy by causing apoptosis and preventing activation and proliferation of T-lymphocytes. In IBD, conventional weight-based dosing with thiopurines leads to an inadequate response in many patients. Utilizing measurement of these metabolites and then employing dose optimization strategies has led to markedly improved outcomes in IBD. Switching between thiopurines as well as the addition of low-dose allopurinol can overcome adverse events and elevate 6TGN levels into the therapeutic window. There is a paucity of data on thiopurine metabolites in rheumatological diseases and further research is required.

Thiopurine pharmacogenomics: association of SNPs with clinical response and functional validation of candidate genes

PubMed Central

Matimba, Alice; Li, Fang; Livshits, Alina; Cartwright, Cher S; Scully, Stephen; Fridley, Brooke L; Jenkins, Gregory; Batzler, Anthony; Wang, Liewei; Weinshilboum, Richard; Lennard, Lynne

2014-01-01

Aim We investigated candidate genes associated with thiopurine metabolism and clinical response in childhood acute lymphoblastic leukemia. Materials & methods We performed genome-wide SNP association studies of 6-thioguanine and 6-mercaptopurine cytotoxicity using lymphoblastoid cell lines. We then genotyped the top SNPs associated with lymphoblastoid cell line cytotoxicity, together with tagSNPs for genes in the ‘thiopurine pathway’ (686 total SNPs), in DNA from 589 Caucasian UK ALL97 patients. Functional validation studies were performed by siRNA knockdown in cancer cell lines. Results SNPs in the thiopurine pathway genes ABCC4, ABCC5, IMPDH1, ITPA, SLC28A3 and XDH, and SNPs located within or near ATP6AP2, FRMD4B, GNG2, KCNMA1 and NME1, were associated with clinical response and measures of thiopurine metabolism. Functional validation showed shifts in cytotoxicity for these genes. Conclusion The clinical response to thiopurines may be regulated by variation in known thiopurine pathway genes and additional novel genes outside of the thiopurine pathway. PMID:24624911

Involvement of MTHFR and TPMT genes in susceptibility to childhood acute lymphoblastic leukemia (ALL) in Mexicans.

PubMed

Gutiérrez-Álvarez, Ossyneidee; Lares-Asseff, Ismael; Galaviz-Hernández, Carlos; Reyes-Espinoza, Elio-Aarón; Almanza-Reyes, Horacio; Sosa-Macías, Martha; Chairez Hernández, Isaías; Salas-Pacheco, José-Manuel; Bailón-Soto, Claudia E

2016-03-01

Folate metabolism plays an essential role in the processes of DNA synthesis and methylation. Deviations in the folate flux resulting from single-nucleotide polymorphisms in genes encoding folate-dependent enzymes may affect the susceptibility to leukemia. This case-control study aimed to assess associations among MTHFR (C677T, A1298C) and TPMT (*2, *3A) mutations as well as to evaluate the synergistic effects of combined genotypes for both genes. Therefore, these genetic variants may lead to childhood acute lymphoblastic leukemia (ALL) susceptibility, in a Mexican population study. DNA samples obtained from 70 children with ALL and 152 age-matched controls (range, 1-15 years) were analyzed by real-time reverse transcription polymerase chain reaction (RT-qPCR) to detect MTHFR C677T and A1298C and TPMT*2 and TPMT*3A genotypes. The frequency of the MTHFR A1298C CC genotype was statistically significant (odds ratio [OR], 6.48; 95% 95% confidence intervals [CI], 1.26-33.2; p=0.025). In addition, the combined 677CC+1298AC genotype exhibited a statistically significant result (OR, 0.23; 95% CI, 0.06-0.82; p=0.023). No significant results were obtained from the MTHFR (C677T CT, C677T TT) or TPMT (*2, *3A) genotypes. More importantly, no association between the synergistic effects of either gene (MTHFR and/or TPMT) and susceptibility to ALL was found. The MTHFR A1298C CC genotype was associated with an increased risk of developing childhood ALL. However, a decreased risk to ALL with the combination of MTHFR 677CC+1298AC genotypes was found.

Metabolite monitoring to guide thiopurine therapy in systemic autoimmune diseases.

PubMed

Chapdelaine, Aurélie; Mansour, Anne-Marie; Troyanov, Yves; Williamson, David R; Doré, Maxime

2017-06-01

6-Thioguanine nucleotide (6-TGN) is the active metabolite of thiopurine drugs azathioprine and 6-mercaptopurine. 6-Methylmercaptopurine (6-MMP) is an inactive and potentially hepatotoxic metabolite. A subgroup of patients (shunters) preferentially produce 6-MMP instead of 6-TGN, therefore displaying thiopurine resistance and risk for hepatotoxicity. Outside inflammatory bowel disease literature, few data exist regarding individualized thiopurine therapy based on metabolite monitoring. This study sought to describe metabolite monitoring in patients receiving weight-based thiopurine for systemic autoimmune diseases. Patients were enrolled using a laboratory database, and data were retrospectively collected. The correlation between the highest thiopurine dose (mg/kg) and the 6-TGN concentration (pmol/8 × 10 8 erythrocytes) was estimated with Pearson's correlation coefficient. Seventy-one patients with various systemic autoimmune conditions were enrolled. The correlation between the thiopurine dose and the 6-TGN level was weak for the overall patient sample (r = 0.201, p = 0.092) and for the subgroup of non-shunters (r = 0.278, p = 0.053). Subjects with 6-MMP levels >5700 pmol/8 × 10 8 erythrocytes had more hepatic cytolysis compared to subjects with 6-MMP <5700, OR = 4.36 (CI 95% 1.18-16.13, p = 0.027). Twenty-two patients (31%) were identified as shunters. Six shunters developed hepatotoxicity, five of which had 6-MMP concentration >5700. Eleven non-shunters had hepatotoxicity, one of which had 6-MMP >5700. Thiopurine metabolite monitoring shows wide variability in 6-TGN levels among patients treated with weight-based thiopurine for systemic autoimmune diseases. Thirty-one percent of the patients in our series fulfilled the shunter definition. Thiopurine metabolite monitoring and dose adjustment to improve maintenance of remission and avoid hepatotoxicity should be studied prospectively.

Replication of TPMT and ABCC3 genetic variants highly associated with cisplatin-induced hearing loss in children.

PubMed

Pussegoda, K; Ross, C J; Visscher, H; Yazdanpanah, M; Brooks, B; Rassekh, S R; Zada, Y F; Dubé, M-P; Carleton, B C; Hayden, M R

2013-08-01

Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. A serious complication of cisplatin treatment is permanent hearing loss. The aim of this study was to replicate previous genetic findings in an independent cohort of 155 pediatric patients. Associations were replicated for genetic variants in TPMT (rs12201199, P = 0.0013, odds ratio (OR) 6.1) and ABCC3 (rs1051640, P = 0.036, OR 1.8). A predictive model combining variants in TPMT, ABCC3, and COMT with clinical variables (patient age, vincristine treatment, germ-cell tumor, and cranial irradiation) significantly improved the prediction of hearing-loss development as compared with using clinical risk factors alone (area under the curve (AUC) 0.786 vs. 0.708, P = 0.00048). The novel combination of genetic and clinical factors predicted the risk of hearing loss with a sensitivity of 50.3% and a specificity of 92.7%. These findings provide evidence to support the importance of TPMT, COMT, and ABCC3 in the prediction of cisplatin-induced hearing loss in children.

Replication of TPMT and ABCC3 Genetic Variants Highly Associated With Cisplatin-Induced Hearing Loss in Children

PubMed Central

Pussegoda, K; Ross, CJ; Visscher, H; Yazdanpanah, M; Brooks, B; Rassekh, SR; Zada, YF; Dubé, M-P; Carleton, BC; Hayden, MR

2014-01-01

Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. A serious complication of cisplatin treatment is permanent hearing loss. The aim of this study was to replicate previous genetic findings in an independent cohort of 155 pediatric patients. Associations were replicated for genetic variants in TPMT (rs12201199, P = 0.0013, odds ratio (OR) 6.1) and ABCC3 (rs1051640, P = 0.036, OR 1.8). A predictive model combining variants in TPMT, ABCC3, and COMT with clinical variables (patient age, vincristine treatment, germ-cell tumor, and cranial irradiation) significantly improved the prediction of hearing-loss development as compared with using clinical risk factors alone (area under the curve (AUC) 0.786 vs. 0.708, P = 0.00048). The novel combination of genetic and clinical factors predicted the risk of hearing loss with a sensitivity of 50.3% and a specificity of 92.7%. These findings provide evidence to support the importance of TPMT, COMT, and ABCC3 in the prediction of cisplatin-induced hearing loss in children. PMID:23588304

Clinical Application of Pharmacogenetics: Where are We Now?

PubMed Central

2014-01-01

Pharmacogenetic (PGx) testing has the potential to improve drug therapy in an individual by informing appropriate drug dosing or drug selection in order to maximize efficacy and safety. Although multiple studies have illustrated the potential benefits of such testing when applied to specific drugs across a broad range of therapy areas, the uptake of PGx testing in routine clinical practice has been relatively limited. Implementation appears to be hampered by the absence of sufficiently strong evidence linking the results of testing with actionable benefits in terms of clinical outcomes. Meanwhile, there are now adequate data to allow dosing recommendations as have been developed by bodies including the Dutch Pharmacogenetics Working Group (DPWG) and the Clinical Pharmacogenetics Implementation Consortium (CPIC) in several settings, including TPMT/thiopurines, CYP2C19/clopidogrel, CYP2D6/codeine, VKORC1-CYP2C9/warfarin, HLA-B*5701/abacavir, SLCO1B1/simvastatin and HLAB*5801/allopurinol. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) have also recently initiated surveys in order to better understand the extent of, and the role played by, PGx testing in clinical practice. This should help identify where further training and education may be beneficial. To this end, in collaboration with ESPT, the IFCC Pharmacogenetic Laboratory Network has now been formed, with the aim of improving the uptake and quality of PGx testing. PMID:27683445

Smoking status and response to thiopurines in steroid-dependent inflammatory bowel disease.

PubMed

Domènech, Eugeni; Carrión, Sílvia; Garcia-Planella, Esther; Mañosa, Míriam; Gordillo, Jordi; Concepción, Mar; Guarner, Carlos; Cabré, Eduard

2011-04-01

The influence of smoking on inflammatory bowel disease (IBD) susceptibility and on its clinical course is well known, but not its impact on drug efficacy. The aim of this study was to evaluate the response to thiopurines in patients with steroid-dependent IBD according to their smoking habits. The medical records of 163 IBD patients (103 Crohn's disease [CD], 60 ulcerative colitis [UC]) in whom thiopurines were started because of steroid-dependency were reviewed. Therapeutic response was defined by steroid-free clinical remission for at least 6 months after 12 months of starting thiopurines. Clinical data and smoking status at diagnosis, at the time thiopurines were started, and during the follow-up were registered. A therapeutic response was obtained in 72% of CD and 61% of UC patients. Smoking habits did not influence the rate of response to thiopurines, the need for rescue therapies, or the development of penetrating/stricturing complications (CD) or proximal progression (UC). However, CD responders who continued smoking required new courses of steroids more often during follow-up. No influence of smoking was found when these outcomes were analyzed depending on gender or disease location. In the multivariate analysis, smoking status was the only predictive factor of drug tolerance. Active smoking does not influence the response to thiopurines in steroid-dependent IBD, but may decrease the likelihood of drug tolerance. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease

PubMed Central

Nguyen, Truc; Vacek, Pamela M.; O’Neill, Patrick; Colletti, Richard B.; Finette, Barry A.

2009-01-01

The thiopurines, azathioprine and 6-mercaptopurine, are effective immune-modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the HPRT locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). Analyses of variance and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment and total therapeutic dose of azathioprine and 6-mercaptopurine. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared to 63 subjects not treated with thiopurines. This increase was related to both total dose (p<0.001) and duration of treatment (p<0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (p <0.001), in particular G:C to A:T transitions (p<0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation and TCRβ CDR3 region unique gene sequence also demonstrated a significant thiopurine-dependent increase in mutant cell clonal proliferation (p<0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention. PMID:19706768

The effects of aminosalicylates or thiopurines on the risk of colorectal cancer in inflammatory bowel disease.

PubMed

Carrat, F; Seksik, P; Colombel, J-F; Peyrin-Biroulet, L; Beaugerie, L

2017-02-01

Whether aminosalicylates or thiopurines reduce the risk of colorectal cancer (CRC) in inflammatory bowel (IBD) disease is controversial. To assess simultaneously the chemopreventive effect of aminosalicylates or thiopurines in a case-control study nested in the CESAME observational cohort that enrolled consecutive patients with IBD between May 2004 and June 2005. Patients were followed up to December 2007. Study population comprised 144 case patients who developed CRC from the diagnosis of IBD (65 and 79 cases diagnosed, respectively, before and from 2004, starting year of the prospective observational period of CESAME) and 286 controls matched for gender, age, IBD subtype and year of diagnosis, and cumulative extent of colitis. Exposure to aminosalicylates or thiopurines was defined by an exposure to the treatment during the year of the diagnosis of cancer. The propensity of receiving 5-ASA and thiopurines was quantified by a composite score taking into account patient and IBD characteristics. The role of aminosalicylates or thiopurines was assessed by multivariate analysis. Propensity scores and the history of primary sclerosing cholangitis were entered into the multivariate model for adjustment. By multivariate analysis adjusted for propensity, a significant protective effect of exposure to drugs during the year of cancer was found for aminosalicylates (OR = 0.587, 95% CI: 0.367-0.937, P = 0.0257), but not for thiopurines (OR = 0.762, 95% CI: 0.432-1.343, P = 0.3468). In a case-control study nested in the CESAME cohort, a significant decrease in the risk of colorectal cancer in IBD was associated with exposure to aminosalicylates, not to thiopurines. © 2016 John Wiley & Sons Ltd.

Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.

PubMed

Abraham, R T; Benson, L M; Jardine, I

1983-10-01

Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.

Optimizing 6-mercaptopurine and azathioprine therapy in the management of inflammatory bowel disease

PubMed Central

Bradford, Kara; Shih, David Q

2011-01-01

The thiopurine drugs, 6-mercaptopurine (6-MP) and azathioprine, are efficacious in the arsenal of inflammatory bowel disease (IBD) therapy. Previous reports indicate that 6-thioguanine nucleotide (6-TGN) levels correlate with therapeutic efficacy, whereas high 6-methylmercaptopurine (6-MMP) levels are associated with hepatotoxicity and myelotoxicity. Due to their complex metabolism, there is wide individual variation in patient response therein, both in achieving therapeutic drug levels as well as in developing adverse reactions. Several strategies to optimize 6-TGN while minimizing 6-MMP levels have been adopted to administer the thiopurine class of drugs to patients who otherwise would not tolerate these drugs due to side-effects. In this report, we will review different approaches to administer the thiopurine medications, including the administration of 6-mercaptopurine in those unsuccessfully treated with azathioprine; co-administration of thiopurine with allopurinol; co-administration of thiopurine with anti-tumor necrosis factor α; 6-TGN administration; desensitization trials; and split dosing of 6-MP. PMID:22072847

Tautomerism and infrared spectra of 2-thiopurine: an experimental matrix isolation and theoretical ab initio and density functional theory study

NASA Astrophysics Data System (ADS)

Stepanenko, Tetyana; Lapinski, Leszek; Nowak, Maciej J.; Kwiatkowski, Józef S.; Leszczynski, Jerzy

2001-02-01

Infrared spectra of 2-thiopurine (2-mercaptopurine, 2-purinethiol ) isolated in low-temperature Ar and N 2 matrixes are reported. These spectra indicate that the compound adopts exclusively the thiol N9H tautomeric form. The theoretical calculations of relative energies of 2-thiopurine tautomers have been carried out at the MP4(SDTQ)//HF level using the 6-31G( d, p) basis set. The thiol N9H tautomer was predicted to be the most stable of all isomers of 2-thiopurine. The infrared spectra of the tautomers of 2-thiopurine have been calculated at the DFT(B3LYP)/6-31G( d, p) level. Good agreement between the experimental spectra and the spectra calculated for thiol N9H tautomer supported the identification of the dominant tautomer. It has also allowed for the reliable assignment of the bands observed in the experimental IR spectrum.

Optimizing 6-mercaptopurine and azathioprine therapy in the management of inflammatory bowel disease.

PubMed

Bradford, Kara; Shih, David Q

2011-10-07

The thiopurine drugs, 6-mercaptopurine (6-MP) and azathioprine, are efficacious in the arsenal of inflammatory bowel disease (IBD) therapy. Previous reports indicate that 6-thioguanine nucleotide (6-TGN) levels correlate with therapeutic efficacy, whereas high 6-methylmercaptopurine (6-MMP) levels are associated with hepatotoxicity and myelotoxicity. Due to their complex metabolism, there is wide individual variation in patient response therein, both in achieving therapeutic drug levels as well as in developing adverse reactions. Several strategies to optimize 6-TGN while minimizing 6-MMP levels have been adopted to administer the thiopurine class of drugs to patients who otherwise would not tolerate these drugs due to side-effects. In this report, we will review different approaches to administer the thiopurine medications, including the administration of 6-mercaptopurine in those unsuccessfully treated with azathioprine; co-administration of thiopurine with allopurinol; co-administration of thiopurine with anti-tumor necrosis factor α; 6-TGN administration; desensitization trials; and split dosing of 6-MP.

Idiopathic portal hypertension regarding thiopurine treatment in patients with inflammatory bowel disease.

PubMed

Suárez Ferrer, Cristina; Llop Herrera, Elba; Calvo Moya, Marta; Vera Mendoza, María Isabel; González Partida, Irene; González Lama, Yago; Matallana Royo, Virginia; Calleja Panero, José Luis; Abreu García, Luis

2016-02-01

The possibility of developing idiopathic portal hypertension has been described with thiopurine treatment despite compromises the prognosis of these patients, the fact its true prevalence is unknown. A cross-sectional study was conducted in a cohort of inflammatory bowel disease (IBD) patients followed at our unit, to determine the prevalence of diagnosis of idiopathic portal hypertension (IPH) and its relationship with thiopurine treatment. At the time of the analysis, 927/1,419 patients were under treatment with thiopurine drugs (65%). A total of 4 patients with IBD type Crohn's disease with idiopathic portal hypertension probably related to the thiopurine treatment were identified (incidence of 4.3 cases per 1,000). Seventy-five percent of patients started with signs or symptoms of portal hypertension. Only one patient was asymptomatic but the diagnosis of IPH because of isolated thrombocytopenia is suspected. However, note that all patients had thrombocytopenia previously. Abdominal ultrasound with fibroscan, hepatic vein catheterization and liver biopsy were performed on all of them as part of the etiology of portal hypertension. In the abdominal ultrasound, indirect portal hypertension data were observed in all patients (as splenomegaly) cirrhosis was also ruled out. The fibroscan data showed significant liver fibrosis (F2-F3). Idiopathic portal hypertension following thiopurine treatment in IBD patients is a rare occurrence, but it must be borne in mind in the differential diagnosis for early diagnosis, especially in patients undergoing thiopurine treatment over a long period. The presence of thrombocytopenia is often the only predictor of its development in the preclinical stage.

Lymphoproliferative disorders in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study.

PubMed

Beaugerie, Laurent; Brousse, Nicole; Bouvier, Anne Marie; Colombel, Jean Frédéric; Lémann, Marc; Cosnes, Jacques; Hébuterne, Xavier; Cortot, Antoine; Bouhnik, Yoram; Gendre, Jean Pierre; Simon, Tabassome; Maynadié, Marc; Hermine, Olivier; Faivre, Jean; Carrat, Fabrice

2009-11-07

Reports of an increased risk of lymphoproliferative disorders in patients receiving thiopurines for inflammatory bowel disease are controversial. We assessed this risk in a prospective observational cohort study. 19,486 patients with inflammatory bowel disease, of whom 11,759 (60.3%) had Crohn's disease and 7727 (39.7%) had ulcerative colitis or unclassified inflammatory bowel disease, were enrolled in a nationwide French cohort by 680 gastroenterologists, who reported details of immunosuppressive therapy during the observation period, cases of cancer, and deaths. The risk of lymphoproliferative disorder was assessed according to thiopurine exposure. Median follow-up was 35 months (IQR 29-40). At baseline, 5867 (30.1%) of patients were receiving, 2809 (14.4%) had discontinued, and 10,810 (55.5%) had never received thiopurines. 23 new cases of lymphoproliferative disorder were diagnosed, consisting of one case of Hodgkin's lymphoma and 22 cases of non-Hodgkin lymphoproliferative disorder. The incidence rates of lymphoproliferative disorder were 0.90 per 1000 (95% CI 0.50-1.49) patient-years in those receiving, 0.20/1000 (0.02-0.72) patient-years in those who had discontinued, and 0.26/1000 (0.10-0.57) patient-years in those who had never received thiopurines (p=0.0054). The multivariate-adjusted hazard ratio of lymphoproliferative disorder between patients receiving thiopurines and those who had never received the drugs was 5.28 (2.01-13.9, p=0.0007). Most cases associated with thiopurine exposure matched the pathological range of post-transplant disease. Patients receiving thiopurines for inflammatory bowel disease have an increased risk of developing lymphoproliferative disorders. Programme Hospitalier de Recherche Clinique National (AOM05157), Association François Aupetit, Délégation Inter-régionale de la Recherche clinique Ile de France-Assistance Publique Hôpitaux de Paris (AP-HP), Ligue contre le Cancer, and Fonds de Recherche de la Société Nationale Française de Gastro-entérologie.

Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells - Effects on gene expression levels and thiopurine metabolism.

PubMed

Haglund, Sofie; Vikingsson, Svante; Almer, Sven; Söderman, Jan

2017-01-01

Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP's effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.

Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells – Effects on gene expression levels and thiopurine metabolism

PubMed Central

Haglund, Sofie; Vikingsson, Svante; Almer, Sven; Söderman, Jan

2017-01-01

Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP’s effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress. PMID:28278299

Pediatric acute lymphoblastic leukemia.

PubMed

Carroll, William L; Bhojwani, Deepa; Min, Dong-Joon; Raetz, Elizabeth; Relling, Mary; Davies, Stella; Downing, James R; Willman, Cheryl L; Reed, John C

2003-01-01

The outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically with current therapy resulting in an event free survival exceeding 75% for most patients. However significant challenges remain including developing better methods to predict which patients can be cured with less toxic treatment and which ones will benefit from augmented therapy. In addition, 25% of patients fail therapy and novel treatments that are focused on undermining specifically the leukemic process are needed urgently. In Section I, Dr. Carroll reviews current approaches to risk classification and proposes a system that incorporates well-established clinical parameters, genetic lesions of the blast as well as early response parameters. He then provides an overview of emerging technologies in genomics and proteomics and how they might lead to more rational, biologically based classification systems. In Section II, Drs. Mary Relling and Stella Davies describe emerging findings that relate to host features that influence outcome, the role of inherited germline variation. They highlight technical breakthroughs in assessing germline differences among patients. Polymorphisms of drug metabolizing genes have been shown to influence toxicity and the best example is the gene thiopurine methyltransferase (TPMT) a key enzyme in the metabolism of 6-mercaptopurine. Polymorphisms are associated with decreased activity that is also associated with increased toxicity. The role of polymorphisms in other genes whose products play an important role in drug metabolism as well as cytokine genes are discussed. In Sections III and IV, Drs. James Downing and Cheryl Willman review their findings using gene expression profiling to classify ALL. Both authors outline challenges in applying this methodology to analysis of clinical samples. Dr. Willman describes her laboratory's examination of infant leukemia and precursor B-ALL where unsupervised approaches have led to the identification of inherent biologic groups not predicted by conventional morphologic, immunophenotypic and cytogenetic variables. Dr. Downing describes his results from a pediatric ALL expression database using over 327 diagnostic samples, with 80% of the dataset consisting of samples from patients treated on a single institutional protocol. Seven distinct leukemia subtypes were identified representing known leukemia subtypes including: BCR-ABL, E2A-PBX1, TEL-AML1, rearrangements in the MLL gene, hyperdiploid karyotype (i.e., > 50 chromosomes), and T-ALL as well as a new leukemia subtype. A subset of genes have been identified whose expression appears to be predictive of outcome but independent verification is needed before this type of analysis can be integrated into treatment assignment. Chemotherapeutic agents kill cancer cells by activating apoptosis, or programmed cell death. In Section V, Dr. John Reed describes major apoptotic pathways and the specific role of key proteins in this response. The expression level of some of these proteins, such as BCL2, BAX, and caspase 3, has been shown to be predictive of ultimate outcome in hematopoietic tumors. New therapeutic approaches that modulate the apoptotic pathway are now available and Dr. Reed highlights those that may be applicable to the treatment of childhood ALL.

Pharmacology of Thiopurine Therapy in Inflammatory Bowel Disease and Complete Blood Cell Count Outcomes: A 5-Year Database Study

PubMed Central

Wilhelm, Abraham J.; Mulder, Chris J. J.; Bouma, Gerd; van Bodegraven, Adriaan A.; de Boer, Nanne K. H.

2017-01-01

Background: Thiopurines are the prerequisite for immunomodulation in inflammatory bowel disease (IBD) therapy. When administered in high (oncological) dose, thiopurine metabolites act as purine antagonists, causing DNA-strand breakage and myelotoxicity. In lower IBD dosages, the mode of action is primarily restricted to anti-inflammatory effects. Then, myelosuppression and hepatotoxicity are the most common adverse events of thiopurines. The aim of this study was to assess the effect of thiopurine metabolites on hematologic and hepatic parameters and to determine which patient characteristics are related to generation of thiopurine metabolites. Methods: The authors scrutinized the therapeutic drug monitoring database of the VU University medical center and subsequently merged this database with the Clinical Laboratory database of our hospital covering the same time period (2010–2015). Results: The authors included 940 laboratory findings of 424 unique patients in this study. Concentrations of 6-thioguanine nucleotides (6-TGN) correlated negatively with red blood cell count, white blood cell count, and neutrophil count in both azathioprine (AZA) and mercaptopurine users. There was a positive correlation with mean corpuscular volume. In patients using 6-thioguanine, 6-TGN concentrations correlated positively with white blood cell count. Furthermore, there was an inverse correlation between patient's age and 6-TGN concentrations in patients using AZA or 6-thioguanine, and we observed an inverse correlation between body mass index and 6-TGN concentrations in patients using AZA or mercaptopurine. No relations were observed with liver test abnormalities. Conclusions: Thiopurine derivative therapy influenced bone marrow production and the size of red blood cells. Age and body mass index were important pharmacokinetic factors in the generation of 6-TGN. PMID:28489727

Thiopurines are associated with a reduction in surgical re-resections in patients with Crohn's disease: a long-term follow-up study in a regional and academic cohort.

PubMed

van Loo, Ellen S; Vosseberg, Ninke W; van der Heide, Frans; Pierie, Jean-Pierre E N; van der Linde, Klaas; Ploeg, Rutger J; Dijkstra, Gerard; Nieuwenhuijs, Vincent B

2013-12-01

Combination therapy of thiopurines and anti-tumor necrosis factor alpha (TNF-α) antibodies is the most effective medical treatment of Crohn's disease (CD). Data on thiopurines and anti-TNF-α antibodies in preventing surgical recurrence (need for re-resection) of CD are scarce. Therefore, we analyzed which factors were involved in surgical recurrence of CD in a large cohort of patients with CD operated in a regional and a university hospital. This is a retrospective cohort study of 567 patients who underwent surgery for CD. Clinical data and risk factors for surgical recurrence were analyzed, focusing on medical therapy and hospital type. Overall, 237 (41.8%) patients developed a surgical recurrence, after a median of 70 (2-482) months. Before surgical recurrence, 235 patients (41.4%) and 116 patients (20.5%) used thiopurines and anti-TNF-α antibodies, respectively. Multivariate analysis identified 3 independent risk factors associated with surgical recurrence of CD. A higher risk was seen in patients with colonic disease compared with patients with ileal disease (hazard ratio, 1.56; 95% confidence interval, 1.10-2.21; P = 0.012) and in patients using multiple types of medication (hazard ratio, 1.38; 95% confidence interval, 1.25-1.54; P < 0.001). However, a lower risk was seen in patients using thiopurines (hazard ratio, 0.51; 95% confidence interval, 0.34-0.77; P = 0.001). Thiopurines are effective in preventing surgical recurrence of CD. The role of anti-TNF-α antibodies seems promising as well. Combination therapy of thiopurines and anti-TNF-α antibodies for prevention of surgical recurrence of CD should be studied in a randomized trial.

Thiopurine treatment in patients with Crohn's disease leads to a selective reduction of an effector cytotoxic gene expression signature revealed by whole-genome expression profiling.

PubMed

Bouma, G; Baggen, J M; van Bodegraven, A A; Mulder, C J J; Kraal, G; Zwiers, A; Horrevoets, A J; van der Pouw Kraan, C T M

2013-07-01

Crohn's disease (CD) is characterized by chronic inflammation of the gastrointestinal tract, as a result of aberrant activation of the innate immune system through TLR stimulation by bacterial products. The conventional immunosuppressive thiopurine derivatives (azathioprine and mercaptopurine) are used to treat CD. The effects of thiopurines on circulating immune cells and TLR responsiveness are unknown. To obtain a global view of affected gene expression of the immune system in CD patients and the treatment effect of thiopurine derivatives, we performed genome-wide transcriptome analysis on whole blood samples from 20 CD patients in remission, of which 10 patients received thiopurine treatment, compared to 16 healthy controls, before and after TLR4 stimulation with LPS. Several immune abnormalities were observed, including increased baseline interferon activity, while baseline expression of ribosomal genes was reduced. After LPS stimulation, CD patients showed reduced cytokine and chemokine expression. None of these effects were related to treatment. Strikingly, only one highly correlated set of 69 genes was affected by treatment, not influenced by LPS stimulation and consisted of genes reminiscent of effector cytotoxic NK cells. The most reduced cytotoxicity-related gene in CD was the cell surface marker CD160. Concordantly, we could demonstrate an in vivo reduction of circulating CD160(+)CD3(-)CD8(-) cells in CD patients after treatment with thiopurine derivatives in an independent cohort. In conclusion, using genome-wide profiling, we identified a disturbed immune activation status in peripheral blood cells from CD patients and a clear treatment effect of thiopurine derivatives selectively affecting effector cytotoxic CD160-positive cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hepatotoxicity associated with 6-methyl mercaptopurine formation during azathioprine and 6-mercaptopurine therapy does not occur on the short-term during 6-thioguanine therapy in IBD treatment.

PubMed

van Asseldonk, Dirk P; Seinen, Margien L; de Boer, Nanne K H; van Bodegraven, Ad A; Mulder, Chris J

2012-02-01

High concentrations of methylated thiopurine metabolites, such as 6-methyl mercaptopurine, are associated with hepatotoxicity during administration of the conventional thiopurines azathioprine or 6-mercaptopurine in IBD patients. Metabolization of the non-conventional thiopurine 6-thioguanine does not generate 6-methyl mercaptopurine. Hence, the aim of our study was to evaluate hepatotoxicity during 6-thioguanine in IBD patients who previously failed conventional thiopurines due to 6-methyl mercaptopurine associated hepatotoxicity. A retrospective single center intercept cohort study was performed of IBD patients using 6-thioguanine between January 2006 and July 2010 after failing conventional thiopurine therapy due to 6-methyl mercaptopurine associated hepatotoxicity. The primary outcome was the occurrence of 6-thioguanine induced hepatotoxicity, scaled according to the Common Terminology Criteria for Adverse Events. Nineteen patients were included. Median duration of 6-thioguanine therapy (median daily dosage 21 mg (9-24)) was 23 weeks (6-96). Hepatotoxicity did not reoccur in 15 out of 19, whereas grade 1 toxicity persisted in 4 patients (p<0.001). Median aspartate aminotransferase and alanine aminotransferase concentrations decreased from 34 U/l (20-59) and 64 U/l (15-175) to 23 U/l (18-40; p=0.003) and 20 U/l (14-48; p=0.019), respectively. Hepatotoxicity does not reoccur during 6-thioguanine treatment in most IBD patients who failed conventional thiopurines due to 6-methyl mercaptopurine associated hepatotoxicity. Hence, at least at short-term, 6-thioguanine appears a justifiable alternative thiopurine for these IBD patients. Copyright © 2011 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

PKCζ and PKMζ are overexpressed in TCF3-rearranged paediatric acute lymphoblastic leukaemia and are associated with increased thiopurine sensitivity.

PubMed

Hartsink-Segers, S A; Beaudoin, J J; Luijendijk, M W J; Exalto, C; Pieters, R; Den Boer, M L

2015-02-01

Both tumour suppressor and oncogenic functions have been ascribed to the atypical zeta isoform of protein kinase C (PKCζ), whereas its constitutively active form PKMζ is almost exclusively expressed in the brain where it has a role in long-term memory. Using primers unique for either isoform, we found that both PKCζ and PKMζ were expressed in a subset of paediatric acute lymphoblastic leukaemia (ALL) cases carrying a TCF3 (E2A) chromosomal rearrangement. Combined PKCζ and PKMζ (PKC/Mζ) protein as well as phosphorylation levels were elevated in ALL cases, especially TCF3-rearranged precursor B-ALL cases, compared with normal bone marrow (P<0.01). Furthermore, high PKC/Mζ expression in primary ALL cells was associated with increased sensitivity to 6-thioguanine and 6-mercaptopurine (P<0.01), thiopurines used in ALL treatment. PKCζ is believed to stabilize mismatch-repair protein MSH2, facilitating thiopurine responsiveness in T-ALL. However, PKC/Mζ knockdown in a TCF3-rearranged cell line model decreased MSH2 expression but did not induce thiopurine resistance, indicative that the link between high PKC/Mζ levels and thiopurine sensitivity in paediatric precursor B-ALL is not directly causal. Collectively, our data indicate that thiopurine treatment may be effective, especially in paediatric TCF3-rearranged ALL and other patients with a high expression of PKC/Mζ.

PKCζ and PKMζ are overexpressed in TCF3-rearranged paediatric acute lymphoblastic leukaemia and are associated with increased thiopurine sensitivity

PubMed Central

Hartsink-Segers, S A; Beaudoin, J J; Luijendijk, M W J; Exalto, C; Pieters, R; Den Boer, M L

2015-01-01

Both tumour suppressor and oncogenic functions have been ascribed to the atypical zeta isoform of protein kinase C (PKCζ), whereas its constitutively active form PKMζ is almost exclusively expressed in the brain where it has a role in long-term memory. Using primers unique for either isoform, we found that both PKCζ and PKMζ were expressed in a subset of paediatric acute lymphoblastic leukaemia (ALL) cases carrying a TCF3 (E2A) chromosomal rearrangement. Combined PKCζ and PKMζ (PKC/Mζ) protein as well as phosphorylation levels were elevated in ALL cases, especially TCF3-rearranged precursor B-ALL cases, compared with normal bone marrow (P<0.01). Furthermore, high PKC/Mζ expression in primary ALL cells was associated with increased sensitivity to 6-thioguanine and 6-mercaptopurine (P<0.01), thiopurines used in ALL treatment. PKCζ is believed to stabilize mismatch-repair protein MSH2, facilitating thiopurine responsiveness in T-ALL. However, PKC/Mζ knockdown in a TCF3-rearranged cell line model decreased MSH2 expression but did not induce thiopurine resistance, indicative that the link between high PKC/Mζ levels and thiopurine sensitivity in paediatric precursor B-ALL is not directly causal. Collectively, our data indicate that thiopurine treatment may be effective, especially in paediatric TCF3-rearranged ALL and other patients with a high expression of PKC/Mζ. PMID:24990612

Analytical Pitfalls of Therapeutic Drug Monitoring of Thiopurines in Patients With Inflammatory Bowel Disease

PubMed Central

Meijer, Berrie; Mulder, Chris J. J.; van Bodegraven, Adriaan A.; de Boer, Nanne K. H.

2017-01-01

Abstract: The use of thiopurines in the treatment of inflammatory bowel disease (IBD) can be optimized by the application of therapeutic drug monitoring. In this procedure, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) metabolites are monitored and related to therapeutic response and adverse events, respectively. Therapeutic drug monitoring of thiopurines, however, is hampered by several analytical limitations resulting in an impaired translation of metabolite levels to clinical outcome in IBD. Thiopurine metabolism is cell specific and requires nucleated cells and particular enzymes for 6-TGN formation. In the current therapeutic drug monitoring, metabolite levels are assessed in erythrocytes, whereas leukocytes are considered the main target cells of these drugs. Furthermore, currently used methods do not distinguish between active nucleotides and their unwanted residual products. Last, there is a lack of a standardized laboratorial procedure for metabolite assessment regarding the substantial instability of erythrocyte 6-TGN. To improve thiopurine therapy in patients with IBD, it is necessary to understand these limitations and recognize the general misconceptions in this procedure. PMID:29040228

Mercaptopurine/Methotrexate Maintenance Therapy of Childhood Acute Lymphoblastic Leukemia: Clinical Facts and Fiction

PubMed Central

Nielsen, Stine N.; Frandsen, Thomas L.; Nersting, Jacob

2014-01-01

The antileukemic mechanisms of 6-mercaptopurine (6MP) and methotrexate (MTX) maintenance therapy are poorly understood, but the benefits of several years of myelosuppressive maintenance therapy for acute lymphoblastic leukemia are well proven. Currently, there is no international consensus on drug dosing. Because of significant interindividual and intraindividual variations in drug disposition and pharmacodynamics, vigorous dose adjustments are needed to obtain a target degree of myelosuppression. As the normal white blood cell counts vary by patients’ ages and ethnicity, and also within age groups, identical white blood cell levels for 2 patients may not reflect the same treatment intensity. Measurements of intracellular levels of cytotoxic metabolites of 6MP and MTX can identify nonadherent patients, but therapeutic target levels remains to be established. A rise in serum aminotransferase levels during maintenance therapy is common and often related to high levels of methylated 6MP metabolites. However, except for episodes of hypoglycemia, serious liver dysfunction is rare, the risk of permanent liver damage is low, and aminotransferase levels usually normalize within a few weeks after discontinuation of therapy. 6MP and MTX dose increments should lead to either leukopenia or a rise in aminotransferases, and if neither is experienced, poor treatment adherence should be considered. The many genetic polymorphisms that determine 6MP and MTX disposition, efficacy, and toxicity have precluded implementation of pharmacogenomics into treatment, the sole exception being dramatic 6MP dose reductions in patients who are homozygous deficient for thiopurine methyltransferase, the enzyme that methylates 6MP and several of its metabolites. In conclusion, maintenance therapy is as important as the more intensive and toxic earlier treatment phases, and often more challenging. Ongoing research address the applicability of drug metabolite measurements for dose adjustments, extensive host genome profiling to understand diversity in treatment efficacy and toxicity, and alternative thiopurine dosing regimens to improve therapy for the individual patient. PMID:24936744

Affinity labeling of a cysteine at or near the catalytic center of Escherichia coli B DNA-dependent RNA polymerase.

PubMed

Miller, J A; Serio, G F; Bear, J L; Howard, R A; Kimball, A P

1980-03-14

9-beta-D-Arabinofuranosyl-6-thiopurine was used to affinity label DNA-dependent RNA polymerase isolated from Escherichia coli B. This substrate analogue displayed competitive type inhibition which could be reversed by addition of a thiol reagent, such as dithiothreitol, while exposure to hydrogen peroxide, a mild oxidizing agent, caused an increase in both the inhibitory and enzyme binding capability of arabinofuranosyl thiopurine. Chromatographic analysis of the products obtained by pronase digestion of the 9-beta-D-arabinofuranosyl-6-[35S]thiopurine-enzyme complex suggests that disulfide bond formation occurs between the inhibitor and a cysteine residue located in or near the active center of the enzyme. In addition, polyacrylamide gel electrophoresis indicated that the arabinofuranosyl thiopurine moeity was bound to the beta' subunit of the enzyme.

Randomised clinical trial: efficacy, safety and dosage of adjunctive allopurinol in azathioprine/mercaptopurine nonresponders (AAA Study).

PubMed

Friedman, A B; Brown, S J; Bampton, P; Barclay, M L; Chung, A; Macrae, F A; McKenzie, J; Reynolds, J; Gibson, P R; Hanauer, S B; Sparrow, M P

2018-04-01

Thiopurine hypermethylation towards 6-methylmercaptopurine (6MMP) instead of 6-thioguanine nucleotides (6TGN) is associated with inefficacy in patients with IBD. Allopurinol reverses such hypermethylation. To prospectively determine efficacy of allopurinol-thiopurine combination and to compare 2 doses of allopurinol. In a multicentre, double-blind trial, patients with clinically active or steroid-dependent IBD and thiopurine shunting were randomised to 50 or 100 mg/d allopurinol and 25% of their screening thiopurine dose, which was subsequently optimised, aiming for 6TGN of 260-500 pmol/8x10 8 RBCs. The primary endpoint was steroid-free clinical remission at 24 weeks. Of 73 patients, 39 (53% [95% CI 42-65]) achieved steroid-free remission, (54% with 50 mg/d and 53% with 100 mg/d). 81% were able to discontinue steroids. Therapeutic 6TGN levels were achieved in both groups. Final thiopurine doses were lower with 100 mg/d allopurinol (P < 0.005). 6MMP: 6TGN ratio decreased from mean 64 to 4 (P < 0.001), being higher with 50 mg/d (6 ± 1.83) than for 100 mg/d ([1 ± 0.16], P = 0.003). Three patients on 50 mg/d failed to sustain low ratios at 24 weeks. Toxicity was minimal; three patients on 50 mg/d allopurinol developed transient leukopenia. Alanine aminotransferase concentrations decreased (P < 0.001) similarly in both arms. Faecal calprotectin levels at study end were lower in patients who achieved the primary endpoint (median 171 [85-541] vs 821[110-5892] ug/g, P = 0.03). Low-dose allopurinol-thiopurine combination safely reverses shunting and optimises 6TGN with associated improvement in disease activity. 100 mg/d allopurinol is preferable due to greater metabolite profile stability and lower thiopurine dose without additional toxicity. © 2018 John Wiley & Sons Ltd.

Increased risk for nonmelanoma skin cancers in patients who receive thiopurines for inflammatory bowel disease.

PubMed

Peyrin-Biroulet, Laurent; Khosrotehrani, Kiarash; Carrat, Fabrice; Bouvier, Anne-Marie; Chevaux, Jean-Baptiste; Simon, Tabassome; Carbonnel, Frank; Colombel, Jean-Frédéric; Dupas, Jean-Louis; Godeberge, Philippe; Hugot, Jean-Pierre; Lémann, Marc; Nahon, Stéphane; Sabaté, Jean-Marc; Tucat, Gilbert; Beaugerie, Laurent

2011-11-01

Patients with inflammatory bowel disease (IBD) who have been exposed to thiopurines might have an increased risk of skin cancer. We assessed this risk among patients in France. We performed a prospective observational cohort study of 19,486 patients with IBD, enrolled from May 2004 to June 2005, who were followed up until December 31, 2007. The incidence of nonmelanoma skin cancer (NMSC) in the general population, used for reference, was determined from the French Network of Cancer Registries. Before the age of 50 years, the crude incidence rates of NMSC among patients currently receiving or who previously received thiopurines were 0.66/1000 and 0.38/1000 patient-years, respectively; these values were 2.59/1000 and 1.96/1000 patient-years for the age group of 50 to 65 years and 4.04/1000 and 5.70/1000 patient-years for patients older than 65 years. Among patients who had never received thiopurines, the incidence of NMSC was zero before the age of 50 years, 0.60/1000 for the ages of 50 to 65 years, and 0.84/1000 for those older than 65 years. A multivariate Cox regression model stratified by propensity score quintiles showed that ongoing thiopurine treatment (hazard ratio [HR], 5.9; 95% confidence interval [CI], 2.1-16.4; P = .0006) and past thiopurine exposure (HR, 3.9; 95% CI, 1.3-12.1; P = .02) were risk factors for NMSC. They also identified age per 1-year increase as a risk factor for NMSC (HR, 1.08; 95% CI, 1.05-1.11; P < .0001). Ongoing and past exposure to thiopurines significantly increases the risk of NMSC in patients with IBD, even before the age of 50 years. These patients should be protected against UV radiation and receive lifelong dermatologic screening. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

NK cells are biologic and biochemical targets of 6-mercaptopurine in Crohn's disease patients.

PubMed

Yusung, Susy; McGovern, Dermot; Lin, Lin; Hommes, Daniel; Lagishetty, Venu; Braun, Jonathan

2017-02-01

NK cells, which contribute to immune defense against certain viral infections and neoplasia, are emerging as modifiers of chronic immunologic diseases including transplant rejection and autoimmune diseases. Immunobiology and genetic studies have implicated NK cells as a modifier of Crohn's disease, a condition often treated with thiopurine agents such as 6-mercaptopurine (6-MP). Here, we demonstrate that thiopurines mediate NK cell apoptosis via a caspase 3 and 9 inclusive pathway, and that this process is triggered by thiopurine-mediated inhibition of Rac1. We also show that CD patients in clinical remission maintained on 6-MP have decreased NK cell Rac1 activity, and decreased NK cell numbers in their intestinal biopsies. These observations suggest that thiopurine targeting of NK cells may be a previously unappreciated therapeutic action of these agents in IBD. Copyright © 2016 Elsevier Inc. All rights reserved.

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers

PubMed Central

Coulthard, Sally A.; Redfern, Christopher P.F.; Vikingsson, Svante; Appell, Malin Lindqvist; Skoglund, Karin; Falk, Ingrid Jakobsen; Hall, Andrew G.; Taylor, Gordon A.; Hogarth, Linda A.

2011-01-01

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) 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. Since the growth of MTAP-deleted tumour 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 leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP−ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs 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. PMID:21282358

Febuxostat as a novel option to optimize thiopurines' metabolism in patients with inadequate metabolite levels.

PubMed

Doré, Maxime; Frenette, Anne Julie; Mansour, Anne-Marie; Troyanov, Yves; Bégin, Josiane

2014-05-01

To report the use of febuxostat in order to potentiate thiopurines' metabolism in a patient on azathioprine (AZA) therapy with low metabolite 6-thioguanine nucleotides (6-TGN) levels and elevated metabolite 6-methylmercaptopurine (6-MMP) levels. A 44-year-old woman with a history of anti-signal recognition particle necrotizing myopathy was treated with AZA-allopurinol combination therapy. When she developed an atypical drug-induced hypersensitivity syndrome, allopurinol was replaced by the new xanthine oxidase (XO) inhibitor febuxostat, at a daily dose of 40 mg. Febuxostat-AZA combination was successful with 6-TGN reaching therapeutic levels while 6-MMP levels remained low. After 5 months, she developed similar manifestations that she had presented on AZA-allopurinol combination. Febuxostat and AZA were then stopped. AZA and 6-MP are both inactive pro-drugs that undergo a complex metabolic transformation leading to active 6-TGN and potentially hepatotoxic 6-MMP. Some patients with unfavorable thiopurine metabolism might benefit from addition of XO inhibitor allopurinol in order to potentiate 6-TGN and reduce 6-MMP levels. It is likely that febuxostat, via its XO inhibition, would exhibit the same effect on thiopurines' metabolism. It has been shown that low dose of febuxostat was able to prevent hypermethylation and to potentiate 6-TGN levels in an AZA-treated patient. Thus, febuxostat could be useful in optimizing thiopurines' metabolism, but more data are needed before this practice can be recommended. The mechanisms by which febuxostat optimizes thiopurines' metabolism remain to be confirmed. Also, the optimal dose of febuxostat for this use remains to be determined.

Thiopurine Drugs Repositioned as Tyrosinase Inhibitors

PubMed Central

Choi, Joonhyeok; Lee, You-Mie; Jee, Jun-Goo

2017-01-01

Drug repositioning is the application of the existing drugs to new uses and has the potential to reduce the time and cost required for the typical drug discovery process. In this study, we repositioned thiopurine drugs used for the treatment of acute leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two successive oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule inhibitors of tyrosinase for therapeutic and cosmetic purposes. Structure-based virtual screening predicted inhibitor candidates from the US Food and Drug Administration (FDA)-approved drugs. Enzyme assays confirmed the thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor with the inhibitory constant of 52 μM. Two other thiopurine drugs, mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibition; mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a poor inhibitor. The inhibitory constant of mercaptopurine (16 μM) was comparable to that of the well-known inhibitor kojic acid (13 μM). The cell-based assay using B16F10 melanoma cells confirmed that the compounds inhibit mammalian tyrosinase. Particularly, 50 μM thioguanine reduced the melanin content by 57%, without apparent cytotoxicity. Cheminformatics showed that the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors. PMID:29283382

Does lymphopenia or macrocytosis reflect 6-thioguanine levels in patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine?

PubMed

Heerasing, N M; Ng, J F; Dowling, D

2016-04-01

The thiopurine drugs, 6-mercaptopurine and azathioprine, remain the mainstay of immunomodulator therapy for inflammatory bowel disease (IBD). Optimal management depends on achieving therapeutic levels of 6-thioguanine (6-TGN), but measuring thiopurine metabolites is associated with significant cost. Thiopurines cause lymphopenia and an increase in mean corpuscular volume (MCV). It is unclear whether any clinically useful correlation exists between 6-TGN levels and lymphocyte count or MCV. The aim of this study is to investigate the correlation between 6-TGN levels and lymphocyte count and MCV in thiopurine-treated patients with IBD. We analysed a prospectively acquired database of 67 patients with IBD treated with thiopurine therapy. The data were analysed looking at the relationship between 6-TGN levels and both lymphocyte count and MCV by using the Spearman's rank correlation coefficient. Twenty-seven (40%) patients had therapeutic 6-TGN levels. Thirty-three (49%) patients had sub-therapeutic 6-TGN levels. A weak positive correlation between 6-TGN levels and lymphocyte count was demonstrated, but this was not statistically significant (Spearman's R = 0.14, P = 0.23). Spearman's rank correlation coefficient between 6-TGN levels and MCV was statistically significant (R = 0.42, P = 0.0005). MCV >101 fL excluded a subtherapeutic 6-TGN level with positive predictive value of 92%. There is no specific lymphopenia that can be assumed to indicate a therapeutic 6-TGN level. The relationship between 6-TGN levels and MCV is likely to be clinically relevant. If MCV is elevated, 6-TGN is unlikely to be sub-therapeutic. MCV is a potential surrogate marker which can rule out sub-therapeutic thiopurine metabolites in patients with IBD treated with azathioprine or 6-mercaptopurine. © 2016 Royal Australasian College of Physicians.

Physiologically based pharmacokinetic model for 6-mercpatopurine: exploring the role of genetic polymorphism in TPMT enzyme activity

PubMed Central

Ogungbenro, Kayode; Aarons, Leon

2015-01-01

Aims To extend the physiologically based pharmacokinetic (PBPK) model developed for 6-mercaptopurine to account for intracellular metabolism and to explore the role of genetic polymorphism in the TPMT enzyme on the pharmacokinetics of 6-mercaptopurine. Methods The developed PBPK model was extended for 6-mercaptopurine to account for intracellular metabolism and genetic polymorphism in TPMT activity. System and drug specific parameters were obtained from the literature or estimated using plasma or intracellular red blood cell concentrations of 6-mercaptopurine and its metabolites. Age-dependent changes in parameters were implemented for scaling, and variability was also introduced for simulation. The model was validated using published data. Results The model was extended successfully. Parameter estimation and model predictions were satisfactory. Prediction of intracellular red blood cell concentrations of 6-thioguanine nucleotide for different TPMT phenotypes (in a clinical study that compared conventional and individualized dosing) showed results that were consistent with observed values and reported incidence of haematopoietic toxicity. Following conventional dosing, the predicted mean concentrations for homozygous and heterozygous variants, respectively, were about 10 times and two times the levels for wild-type. However, following individualized dosing, the mean concentration was around the same level for the three phenotypes despite different doses. Conclusions The developed PBPK model has been extended for 6-mercaptopurine and can be used to predict plasma 6-mercaptopurine and tissue concentration of 6-mercaptopurine, 6-thioguanine nucleotide and 6-methylmercaptopurine ribonucleotide in adults and children. Predictions of reported data from clinical studies showed satisfactory results. The model may help to improve 6-mercaptopurine dosing, achieve better clinical outcome and reduce toxicity. PMID:25614061

Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes.

PubMed

Petit, Elise; Langouet, Sophie; Akhdar, Hanane; Nicolas-Nicolaz, Christophe; Guillouzo, André; Morel, Fabrice

2008-04-01

Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are therapeutic compounds widely administered in the clinic for their multiple uses (autoimmune diseases, post-transplant immunosuppression and cancer). Despite these advantages, their therapeutic potential is limited by occasional adverse effects (myelotoxicity and hepatotoxicity) and by a relatively frequent lack of efficacy. Previous studies have demonstrated that azathioprine decreased the viability of rat hepatocytes. In order to investigate cytotoxic effects of thiopurines in human liver, we used primary human hepatocytes and a highly differentiated human hepatoma cell line, HepaRG, treated or not with azathioprine, 6-mercaptopurine and 6-thioguanine. In parallel, expression of the genes involved in the metabolism of thiopurines, glutathione synthesis and antioxidant defences was measured by quantitative PCR. We clearly demonstrate that human liver parenchymal cells were much less sensitive than rat hepatocytes to thiopurine treatments. The toxic effects appeared after 96 h of treatment while ATP depletion was observed after a 24 h incubation with azathioprine and 6-mercaptopurine. Toxic effects were more pronounced for azathioprine and 6-mercaptopurine, when compared to 6-thioguanine, and might explain glutathione synthesis and antioxidant enzyme induction only by these two drugs. Finally, we also demonstrate for the first time an up-regulation by azathioprine and 6-mercaptopurine of inosine monophosphate dehydrogenase which might have consequences on the de novo biosynthesis of guanine nucleotides and thiopurines metabolism.

Thiopurines in inflammatory bowel disease revisited

PubMed Central

Bär, Florian; Sina, Christian; Fellermann, Klaus

2013-01-01

Although a great variety of new drugs have been introduced for the therapy of inflammatory bowel diseases so far, a definite cure of the disease is still out of scope. An anti-inflammatory approach to induce remission followed by maintenance therapy with immunosupressants is still the mainstay of therapy. Thiopurines comprising azathioprine and its active metabolite mercaptopurine as well as tioguanine, are widely used in the therapy of chronic active inflammatory bowel disease (IBD). Their steroid sparing potential and efficacy in remission maintenance are out of doubt. Unfortunately, untoward adverse events are frequently observed and may preclude further administration or be life threatening. This review will focus on new aspects of thiopurine therapy in IBD, its efficacy and safety. PMID:23555158

Limited stability of thiopurine metabolites in blood samples: relevant in research and clinical practise.

PubMed

de Graaf, P; Vos, R M; de Boer, N H K; Sinjewel, A; Jharap, B; Mulder, C J J; van Bodegraven, A A; Veldkamp, A I

2010-06-01

Monitoring of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) is used to assess compliance and explain adverse reactions in IBD-patients. Correlations between dosage, metabolite concentrations and therapeutic efficacy or toxicity are contradictive. Research is complicated by analytical problems as matrices analyzed and analytical procedures vary widely. Moreover, stability of thiopurine metabolites is not well documented, yet pivotal for interpretation of analytical outcomes. Therefore, we prospectively investigated metabolite stability in blood samples under standard storage conditions. Stability at room temperature and refrigeration (22 degrees C, 4 degrees C) was investigated during 1 week and frozen samples (-20 degrees C, -80 degrees C) were analyzed during 6 months storage. Ten patient samples were analyzed for each study period. Median 6-TGN concentrations on day 7 decreased significantly to 53% and 90% during storage at ambient temperature or refrigeration. Median 6-MMP concentrations on day 7 decreased significantly to 55% and 86%, respectively. Samples stored at -20 degrees C also showed significant decreases in both 6-TGN and 6-MMP in comparison with baseline values. At -80 degrees C, only 6-MMP showed a significant decrease in values compared to baseline. The stability of thiopurine metabolites is clearly a limiting factor in studies investigating utilisation of TDM and correlations with therapeutic outcome in IBD-patients. This has to be accounted for in clinical practice and (multi-center) trials investigating thiopurine drugs. Copyright 2010. Published by Elsevier B.V.

Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs.

PubMed

Eklund, Birgitta I; Gunnarsdottir, Sjofn; Elfarra, Adnan A; Mannervik, Bengt

2007-06-01

cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an alpha,beta-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.

Placental involvement by non-Hodgkin lymphoma in a Crohn disease patient on long-term thiopurine therapy.

PubMed

Chen, G; Crispin, P; Cherian, M; Dahlstrom, J E; Sethna, F F; Kaye, G; Pavli, P; Subramaniam, K

2016-01-01

We report the first published case of aggressive diffuse large B-cell (non-Hodgkin) lymphoma in a 35-year-old pregnant woman who had Crohn disease and was taking long-term thiopurine therapy: the patient developed placental insufficiency, and there was intrauterine fetal death. © 2015 Royal Australasian College of Physicians.

Monitoring for Extra-Intestinal Cancers in IBD.

PubMed

Sifuentes, H; Kane, S

2015-11-01

Multiple studies have demonstrated an increased risk for extra-intestinal cancers in inflammatory bowel disease (IBD) patients, mainly from treatment modalities. Prominent cancers that are related to IBD treatment include the following: lymphoproliferative disorders associated with thiopurine use, hepatosplenic T cell lymphoma primarily in younger male patients on thiopurines and anti-tumor necrosis factor (TNF) agents, non-melanoma skin cancers in patients treated with thiopurines and anti-TNF agents, and melanomas in patients who are on monotherapy with anti-TNF agents. In addition, women with IBD may have higher rates of cervical dysplasia and cervical cancer. The focus of this review is to provide a comprehensive overview on extra-intestinal cancers in IBD patients and how to monitor for these malignancies.

Small intestinal bacterial overgrowth in inactive Crohn's disease: influence of thiopurine and biological treatment.

PubMed

Sánchez-Montes, Cristina; Ortiz, Vicente; Bastida, Guillermo; Rodríguez, Ester; Yago, María; Beltrán, Belén; Aguas, Mariam; Iborra, Marisa; Garrigues, Vicente; Ponce, Julio; Nos, Pilar

2014-10-14

To investigate the influence of thiopurines and biological drugs on the presence of small intestinal bacterial overgrowth (SIBO) in patients with inactive Crohn's disease (CD). This was a prospective study in patients with CD in remission and without corticosteroid treatment, included consecutively from 2004 to 2010. SIBO was investigated using the hydrogen glucose breath test. One hundred and seven patients with CD in remission were included. Almost 58% of patients used maintenance immunosuppressant therapy and 19.6% used biological therapy. The prevalence of SIBO was 16.8%. No association was observed between SIBO and the use of thiopurine Immunosuppressant (12/62 patients), administration of biological drugs (2/21 patients), or with double treatment with an anti-tumor necrosis factor drugs plus thiopurine (1/13 patients). Half of the patients had symptoms that were suggestive of SIBO, though meteorism was the only symptom that was significantly associated with the presence of SIBO on univariate analysis (P < 0.05). Multivariate analysis revealed that the presence of meteorism and a fistulizing pattern were associated with the presence of SIBO (P < 0.05). Immunosuppressants and/or biological drugs do not induce SIBO in inactive CD. Fistulizing disease pattern and meteorism are associated with SIBO.

Pregnancy outcomes in women with inflammatory bowel disease following exposure to thiopurines and antitumor necrosis factor drugs: a systematic review with meta-analysis.

PubMed

Mozaffari, S; Abdolghaffari, A H; Nikfar, S; Abdollahi, M

2015-05-01

Several studies have indicated the harmful effect of flare-up periods in pregnant women with inflammatory bowel disease (IBD) on their newborns. Therefore, an effective and safe medical treatment during pregnancy is of great concern in IBD patients. The aim of this study was to perform a meta-analysis on the outcomes of thiopurines use and a systematic review of antitumor necrosis factor (anti-TNF) drugs used during pregnancy in women with IBD. The results of cohorts evaluating the safety of anti-TNF drugs during pregnancy up to July 2013 were collected and analyzed. In the meta-analysis, a total of 312 pregnant women with IBD who used thiopurines were compared with 1149 controls (women with IBD who were not treated with any medication and women who were exposed to drugs other than thiopurines) to evaluate the drug effect on different pregnancy outcomes, including prematurity, low birth weight, congenital abnormalities, spontaneous abortion, and neonatal adverse outcomes. Results of statistical analysis demonstrated that congenital abnormalities were increased significantly in thiopurine-exposed group in comparison with control group who did not receive any medicine for IBD treatment. The summary odds ratio was 2.95 with 95% confidence interval = 1.03-8.43 (p = 0.04). We observed no significant differences in occurrence of other adverse pregnancy outcomes between compared groups. The results of cohorts evaluated the safety of anti-TNF drugs during pregnancy demonstrated no increase in occurrence of adverse pregnancy outcomes in comparison with controls except for the significant decrease in gestational age of newborns of drug-exposed mothers in one trial. In conclusion, a benefit-risk ratio should be considered in prescribing or continuing medicinal therapy during pregnancy of IBD patients. © The Author(s) 2014.

Small intestinal bacterial overgrowth in inactive Crohn’s disease: Influence of thiopurine and biological treatment

PubMed Central

Sánchez-Montes, Cristina; Ortiz, Vicente; Bastida, Guillermo; Rodríguez, Ester; Yago, María; Beltrán, Belén; Aguas, Mariam; Iborra, Marisa; Garrigues, Vicente; Ponce, Julio; Nos, Pilar

2014-01-01

AIM: To investigate the influence of thiopurines and biological drugs on the presence of small intestinal bacterial overgrowth (SIBO) in patients with inactive Crohn’s disease (CD). METHODS: This was a prospective study in patients with CD in remission and without corticosteroid treatment, included consecutively from 2004 to 2010. SIBO was investigated using the hydrogen glucose breath test. RESULTS: One hundred and seven patients with CD in remission were included. Almost 58% of patients used maintenance immunosuppressant therapy and 19.6% used biological therapy. The prevalence of SIBO was 16.8%. No association was observed between SIBO and the use of thiopurine Immunosuppressant (12/62 patients), administration of biological drugs (2/21 patients), or with double treatment with an anti-tumor necrosis factor drugs plus thiopurine (1/13 patients). Half of the patients had symptoms that were suggestive of SIBO, though meteorism was the only symptom that was significantly associated with the presence of SIBO on univariate analysis (P < 0.05). Multivariate analysis revealed that the presence of meteorism and a fistulizing pattern were associated with the presence of SIBO (P < 0.05). CONCLUSION: Immunosuppressants and/or biological drugs do not induce SIBO in inactive CD. Fistulizing disease pattern and meteorism are associated with SIBO. PMID:25320539

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death

PubMed Central

Morgan, Jessica A.; Lynch, John; Panetta, John C.; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T.; Janke, Laura; Green, Daniel M.; Chemaitilly, Wassim; Schuetz, John D.

2015-01-01

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer. PMID:26576726

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death.

PubMed

Morgan, Jessica A; Lynch, John; Panetta, John C; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T; Janke, Laura; Green, Daniel M; Chemaitilly, Wassim; Schuetz, John D

2015-11-18

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer.

Optimization of the treatment with immunosuppressants and biologics in inflammatory bowel disease

PubMed Central

Renna, Sara; Cottone, Mario; Orlando, Ambrogio

2014-01-01

Many placebo controlled trials and meta-analyses evaluated the efficacy of different drugs for the treatment of inflammatory bowel disease (IBD), including immunosuppressants and biologics. Their use is indicated in moderate to severe disease in non responders to corticosteroids and in steroid-dependent patients, as induction and maintainance treatment. Infliximab, as well as cyclosporine, is considered a second line therapy in the case of severe ulcerative colitis, or non-responders to intravenous corticosteroids. An adequate dosage and duration of therapy with thiopurines should be reached before evaluating their efficacy. Methotrexate is a valid option in patients with Crohn’s disease but its use is confined to patients who are intolerant or non-responders to thiopurines. Evidence for the use of methotrexate in ulcerative colitis is insufficient. The use of thalidomide and mycophenolate mofetil is not recommended in patients with inflammatory bowel disease, these treatments could be considered in case of failure of all other therapeutic options. In patients with moderately active ulcerative colitis, refractory to thiopurines, the use of tacrolimus is considered an alternative to biologics. An increase of the dose or a decrease in the interval of administration of biological treatment could be useful in the presence of an incomplete clinical response. In the case of primary failure of an anti-tumor necrosis factor alpha a switch to another one should be considered. Data on the efficacy of combination therapy are up to now insufficient to consider this strategy in all IBD patients. The final outcome of the treatment should be considered the clinical remission, with mucosa healing, and not the clinical response. The evaluation of serum concentration of thiopurine methyl transferase activity, thiopurine metabolites, biologic serum levels and antibiologic antibodies could be useful for the management of the treatment but it has not been routinely applied in clinical practice. The evidence of high risk development of lymphoma and cutaneous malignancies should be considered in patients treated with immunosuppressants and biologics for a long period. PMID:25110407

NUDT15 codon 139 is the best pharmacogenetic marker for predicting thiopurine-induced severe adverse events in Japanese patients with inflammatory bowel disease: a multicenter study.

PubMed

Kakuta, Yoichi; Kawai, Yosuke; Okamoto, Daisuke; Takagawa, Tetsuya; Ikeya, Kentaro; Sakuraba, Hirotake; Nishida, Atsushi; Nakagawa, Shoko; Miura, Miki; Toyonaga, Takahiko; Onodera, Kei; Shinozaki, Masaru; Ishiguro, Yoh; Mizuno, Shinta; Takahara, Masahiro; Yanai, Shunichi; Hokari, Ryota; Nakagawa, Tomoo; Araki, Hiroshi; Motoya, Satoshi; Naito, Takeo; Moroi, Rintaro; Shiga, Hisashi; Endo, Katsuya; Kobayashi, Taku; Naganuma, Makoto; Hiraoka, Sakiko; Matsumoto, Takayuki; Nakamura, Shiro; Nakase, Hiroshi; Hisamatsu, Tadakazu; Sasaki, Makoto; Hanai, Hiroyuki; Andoh, Akira; Nagasaki, Masao; Kinouchi, Yoshitaka; Shimosegawa, Tooru; Masamune, Atsushi; Suzuki, Yasuo

2018-06-19

Despite NUDT15 variants showing significant association with thiopurine-induced adverse events (AEs) in Asians, it remains unclear which variants of NUDT15 or whether additional genetic variants should be tested to predict AEs. To clarify the best pharmacogenetic test to be used clinically, we performed association studies of NUDT15 variants and haplotypes with AEs, genome-wide association study (GWAS) to discover additional variants, and ROC analysis to select the model to predict severe AEs. Overall, 2630 patients with inflammatory bowel disease (IBD) were enrolled and genotyped for NUDT15 codon 139; 1291 patients were treated with thiopurines. diplotypes were analyzed in 970 patients, and GWASs of AEs were performed with 1221 patients using population-optimized genotyping array and imputation. We confirmed the association of NUDT15 p.Arg139Cys with leukopenia and alopecia (p = 2.20E-63, 1.32E-69, OR = 6.59, 12.1, respectively), and found a novel association with digestive symptoms (p = 6.39E-04, OR = 1.89). Time to leukopenia was significantly shorter, and when leukopenia was diagnosed, thiopurine doses were significantly lower in Arg/Cys and Cys/Cys than in Arg/Arg. In GWASs, no additional variants were found to be associated with thiopurine-induced AEs. Despite strong correlation of leukopenia frequency with estimated enzyme activities based on the diplotypes (r 2  = 0.926, p = 0.0087), there were no significant differences in the AUCs of diplotypes from those of codon 139 to predict severe AEs (AUC = 0.916, 0.921, for acute severe leukopenia, AUC = 0.990, 0.991, for severe alopecia, respectively). Genotyping of NUDT15 codon 139 was sufficient to predict acute severe leukopenia and alopecia in Japanese patients with IBD.

Advances of molecular clinical pharmacology in gastroenterology and hepatology.

PubMed

Prandota, Joseph

2010-01-01

During developmental age, differences in pharmacodynamic reactions to several drugs may reflect polymorphisms of genes encoding drug-transporting proteins, receptors, drug targets, and gene products, whose disturbed activity sometimes plays an important role in certain diseases. Administration of drugs with a narrow therapeutic index may quite easily be associated with changes in pharmacokinetics and development of adverse drug reactions, which occasionally may cause fatalities. In such cases, polypragmasy and resulting drug interactions may enhance effects of changes in drug-metabolizing enzymes' activities. Phenotyping and genotyping of patients slowly are finding their place in some therapeutic regimens used in clinical gastroenterology and hepatology. At present, some assays to measure, for example, thiopurine S-methyltransferase activity are already commercially available. Polymorphisms of CYP450 enzymes, interleukins, and altered gene expression play an important role in some patients' various gastrointestinal tract and liver diseases. Herbal drugs also affect proinflammatory and antiinflammatory cytokine and nitric oxide balance in the body. Therapeutic use of recombined proteins, such as infliximab, natalizumab, onercept, humanized antibody to integrin α-4 β-7, or IFN-β in some large-bowel diseases increased therapeutic efficacy. IFN-α used in the patients with chronic hepatitis C improved cellular immunity in these subjects and exerted antiviral activity. Practical application of progress in pharmacogenetics, pharmacokinetics, pharmacodynamics, and use of bioproducts in novel therapeutic regimens has opened therapeutic frontiers and increased clinical safety.

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF-GAP suggests that host vesicle trafficking is a fungal pathogenicity target.

PubMed

Schmidt, Sarah M; Kuhn, Hannah; Micali, Cristina; Liller, Corinna; Kwaaitaal, Mark; Panstruga, Ralph

2014-08-01

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking. © 2013 BSPP AND JOHN WILEY & SONS LTD.

[No best treatment for severe outbreaks: Maintenance, the key in colitis].

PubMed

Sicilia, Beatriz

2011-12-01

Several drugs are currently available to maintain remission in patients who have responded after one or other type of induction therapy, depending on the initial severity of the outbreak. Salicylates are the drugs of choice to maintain remission after a mild-to-moderate outbreak controlled by salicylates or oral corticosteroids. To maintain remission after a severe outbreak or in patients with corticosteroid dependence or resistance, thiopurines are the drugs of choice. In patients who have failed to respond to thiopurines and in those with thiopurine intolerance, biological agents, mainly infliximab, can be used to maintain remission in patients after induction therapy with infliximab for a severe outbreak. However, these scenarios may not reflect reality of gastroenterologists' daily clinical practice. Treatment will therefore be based on the patient's individual characteristics (age, clinical course, previous treatment, adverse effects and personal preferences) as well as the physician's medical art. Copyright © 2011 Elsevier España, S.L.U. All rights reserved.

Anti-TNF and thiopurine therapy in pregnant IBD patients does not significantly alter a panel of B-cell and T-cell subsets in 1-year-old infants.

PubMed

Kattah, Michael G; Milush, Jeffrey M; Burt, Trevor; McCabe, Robert P; Whang, Michael I; Ma, Averil; Mahadevan, Uma

2018-04-03

Infants exposed to combination therapy with anti-tumor necrosis factor (anti-TNF) agents and thiopurines may exhibit increased infections at 1 year of age compared to unexposed infants. We hypothesized that this increased risk of infection is due to abnormal development of the newborn immune system. We immunophenotyped B-cell and T-cell subsets using multiparameter flow cytometry in 1-year-old infants whose mothers were exposed to therapeutic agents for IBD. We analyzed samples from infants exposed to infliximab (IFX) or adalimumab (ADA) monotherapy (IFX/ADA, n = 11), certolizumab pegol (CZP) monotherapy (CZP, n = 4), IFX or ADA plus thiopurine combination therapy (IFX/ADA + IM, n = 4), and CZP plus thiopurine combination therapy (CZP + IM, n = 2). Percentages of B cells, CD4 + T helper cells, T regulatory cells (T regs ), and CD8 + cytotoxic T cells, were similar among the groups. Infants exposed to combination therapy (IFX/ADA + IM) exhibited trends toward fewer CD27 + B cells, switched memory B cells, plasmablasts, interferon gamma (IFNγ)-producing CD4 + and CD8 + T cells, and CCR5 + CD4 + T cells, but these did not reach statistical significance. Multiparameter immunophenotyping of major B-cell and T-cell subsets suggests that the adaptive newborn immune system develops largely unaltered after exposure to combination therapy as compared to anti-TNF monotherapy.

Clinical Value of Mercaptopurine After Failing Azathioprine Therapy in Patients With Inflammatory Bowel Disease.

PubMed

Meijer, Berrie; Seinen, Margien L; Leijte, Niek N W; Mulder, Chris J J; van Bodegraven, Adriaan A; de Boer, Nanne K H

2016-08-01

Thiopurines have been widely accepted as immunosuppressive therapy in inflammatory bowel disease. However, many patients have to discontinue thiopurines due to intolerance or ineffectiveness. A therapeutically beneficial effect of switching from azathioprine (AZA) to mercaptopurine (MP) after developing adverse events (AEs) has been reported. The authors assessed the clinical value of MP therapy after AZA discontinuation due to intolerance and, secondary, due to ineffectiveness. In this retrospective single-center study, the authors analyzed data from patients in whom AZA therapy had failed and who were subsequently treated with MP. Thirty-eight patients initiated MP therapy after intolerance to AZA. Intolerance reoccurred in 22 (58%) patients and the remaining 16 (42%) tolerated MP. In 18 out of 48 patients (38%), the AEs that led to discontinuation of MP were similar to those of AZA. A longer duration of prior AZA use was more common in patients who were subsequently tolerant for MP (5.3 versus 1.2 months; P = 0.04). Twenty-two patients discontinued AZA due to ineffectiveness. Eight (36%) patients had clinical benefit from a switch to MP. Six out of these 8 (75%) patients used allopurinol alongside MP, due to ineffectiveness based on a skewed thiopurine metabolism. Patients were more likely to have clinical benefit if the interval between both thiopurines was longer (4.4 versus 0.01 months; P < 0.05). The authors showed that a noteworthy number of patients benefitted therapeutically from a switch from AZA to MP when failing due to intolerance or ineffectiveness; however, the percentage was lower than previously reported in literature.

Tolerability profile of thiopurines in inflammatory bowel disease: a prospective experience.

PubMed

Macaluso, Fabio Salvatore; Renna, Sara; Maida, Marcello; Dimarco, Mariangela; Sapienza, Chiara; Affronti, Marco; Orlando, Emanuele; Rizzuto, Giulia; Orlando, Rosalba; Ventimiglia, Marco; Cottone, Mario; Orlando, Ambrogio

2017-09-01

The occurrence of thiopurine-related adverse events (AEs) may complicate the management of patients with inflammatory bowel disease (IBD). We aimed to evaluate the tolerability of thiopurines in a current IBD setting. All consecutive patients who started a treatment with azathioprine (AZA) from January 2010 to March 2016 were entered in a prospectively maintained database, and the AEs which led to the permanent discontinuation of the drug were reported. Two hundred and fifty three patients were included. Median total follow-up was 32 months (range: 0.2-75 months). At the end of the study, AZA was discontinued in 160 patients (63.2%). The main reason leading to drug withdrawal was the occurrence of AEs (109/160 patients [68.1%]; cumulative incidence among the entire cohort: 43.1%). Overall, the most frequent AEs leading to treatment withdrawal were nausea (31/253 patients, 12.3%) and subjective symptoms, i.e., poorly defined side effects such as fatigue, headache and muscle pain (20/253 patients, 7.9%). Among the 109 AZA-intolerant patients, a switch to 6-mercaptopurine (6-MP) was performed in 44 cases (40.4%). At the end of follow-up, 6-MP was discontinued in 35/44 patients (79.5%), mostly due to AEs (29/35 patients, 82.8%). Azathioprine-induced hepatic and pancreatic toxicity was associated with male gender (p = .01 and p = .03, respectively), and occurrence of nausea with Crohn's disease (p = .04). Our real-life prospective cohort showed the higher cumulative incidence of thiopurine withdrawal due to AEs reported to date. Switching from AZA to 6-MP was often ineffective.

Cost-utility Analysis: Thiopurines Plus Endoscopy-guided Biological Step-up Therapy is the Optimal Management of Postoperative Crohn's Disease.

PubMed

Candia, Roberto; Naimark, David; Sander, Beate; Nguyen, Geoffrey C

2017-11-01

Postoperative recurrence of Crohn's disease is common. This study sought to assess whether the postoperative management should be based on biological therapy alone or combined with thiopurines and whether the therapy should be started immediately after surgery or guided by either endoscopic or clinical recurrence. A Markov model was developed to estimate expected health outcomes in quality-adjusted life years (QALYs) and costs in Canadian dollars (CAD$) accrued by hypothetical patients with high recurrence risk after ileocolic resection. Eight strategies of postoperative management were evaluated. A lifetime time horizon, an annual discount rate of 5%, a societal perspective, and a cost-effectiveness threshold of 50,000 CAD$/QALY were assumed. Deterministic and probabilistic sensitivity analyses were conducted. The model was validated against randomized trials and historical cohorts. Three strategies dominated the others: endoscopy-guided full step-up therapy (14.80 QALYs, CAD$ 462,180), thiopurines immediately post-surgery plus endoscopy-guided biological step-up therapy (14.89 QALYs, CAD$ 464,099) and combination therapy immediately post-surgery (14.94 QALYs, CAD$ 483,685). The second strategy was the most cost-effective, assuming a cost-effectiveness threshold of 50,000 CAD$/QALY. Probabilistic sensitivity analysis showed that the second strategy has the highest probability of being the optimal alternative in all comparisons at cost-effectiveness thresholds from 30,000 to 100,000 CAD$/QALY. The strategies guided only by clinical recurrence and those using biologics alone were dominated. According to this decision analysis, thiopurines immediately after surgery and addition of biologics guided by endoscopic recurrence is the optimal strategy of postoperative management in patients with Crohn's disease with high risk of recurrence (see Video Abstract, Supplemental Digital Content 1, http://links.lww.com/IBD/B654).

Azathioprine and 6-Mercaptopurine-induced Liver Injury: Clinical Features and Outcomes.

PubMed

Björnsson, Einar S; Gu, Jiezhun; Kleiner, David E; Chalasani, Naga; Hayashi, Paul H; Hoofnagle, Jay H

2017-01-01

The objective of the study was to define the clinical, biochemical, and histologic features of liver injury from thiopurines. Azathioprine (Aza) and 6-mercaptopurine (6-MP) can cause liver injury, but no large series exist. Clinical and laboratory data and 6-month outcomes of patients with thiopurine hepatotoxicity from the Drug-Induced Liver Injury Network Prospective Study were analyzed. Twenty-two patients were identified, 12 due to Aza and 10 due to 6-MP, with a median age of 55 years; the majority were female (68%). Inflammatory bowel disease was the indication in 55%, and the median thiopurine dose was 150 (range, 25 to 300) mg daily. The median latency to onset was 75 (range, 3 to 2584) days. Injury first arose after a dose escalation in 59% of patients, the median latency after dose increase being 44 (range, 3 to 254) days. At onset, the median alanine aminotransferase level was 210 U/L, alkaline phosphatase was 151 U/L, and bilirubin was 7.4 mg/dL (peak, 13.4 mg/dL). There were no major differences between Aza and 6-MP cases, but anicteric cases typically had nonspecific symptoms and a hepatocellular pattern of enzyme elevations, whereas icteric cases experienced cholestatic hepatitis with modest enzyme elevations in a mixed pattern. One patient with preexisting cirrhosis required liver transplantation; all others resolved clinically. One patient still had moderate alkaline phosphatase elevations 2 years after onset. Nearly three-quarters of patients with thiopurine-induced liver injury present with self-limited, cholestatic hepatitis, typically within 3 months of starting or a dose increase. The prognosis is favorable except in patients with preexisting cirrhosis.

Azathioprine and 6-Mercaptopurine Induced Liver Injury: Clinical Features and Outcomes

PubMed Central

Björnsson, Einar S.; Gu, Jiezhun; Kleiner, David E.; Chalasani, Naga; Hayashi, Paul H.; Hoofnagle, Jay H.

2017-01-01

Goals To define the clinical, biochemical and histologic features of liver injury from thiopurines. Background Azathioprine (Aza) and 6-mercaptopurine (6-MP) can cause liver injury but no large series exist. Methods Clinical and laboratory data and 6-months outcomes were analyzed from patients with thiopurine hepatotoxicity from the Drug-Induced Liver Injury Network Prospective Study. Results 22 patients were identified, 12 due to Aza and 10 6-MP, with a median age of 55 years and the majority females (68%). Inflammatory bowel disease was the indication in 55%, and median thiopurine dose 150 (range 25–300) mg daily. The median latency to onset was 75 (range 3 to 2584) days. Injury first arose after a dose escalation in 59% of patients; the median latency after dose increase being 44 (range 3 to 254) days. At onset, the median alanine aminotransferase was 210 U/L, alkaline phosphatase 151 U/L and bilirubin 7.4 mg/dL (peak 13.4 mg/dL). There were no major differences between Aza and 6-MP cases, but anicteric cases typically had non-specific symptoms and a hepatocellular pattern of enzyme elevations, whereas icteric cases experienced a cholestatic hepatitis with modest enzyme elevations in a mixed pattern. One patient with pre-existing cirrhosis required liver transplantation, all others resolved clinically. One patient still had moderate alkaline phosphatase elevations 2 years after onset. Conclusions Nearly three-quarters of patients with thiopurine-induced liver injury present with self-limited, cholestatic hepatitis, typically within 3 months of starting or a dose increase. The prognosis is favorable except in patients with pre-existing cirrhosis. PMID:27648552

Genetic variants in TPMT and COMT are associated with hearing loss in children receiving cisplatin chemotherapy.

PubMed

Ross, Colin J D; Katzov-Eckert, Hagit; Dubé, Marie-Pierre; Brooks, Beth; Rassekh, S Rod; Barhdadi, Amina; Feroz-Zada, Yassamin; Visscher, Henk; Brown, Andrew M K; Rieder, Michael J; Rogers, Paul C; Phillips, Michael S; Carleton, Bruce C; Hayden, Michael R

2009-12-01

Cisplatin is a widely used and effective chemotherapeutic agent, although its use is restricted by the high incidence of irreversible ototoxicity associated with it. In children, cisplatin ototoxicity is a serious and pervasive problem, affecting more than 60% of those receiving cisplatin and compromising language and cognitive development. Candidate gene studies have previously reported associations of cisplatin ototoxicity with genetic variants in the genes encoding glutathione S-transferases and megalin. We report association analyses for 220 drug-metabolism genes in genetic susceptibility to cisplatin-induced hearing loss in children. We genotyped 1,949 SNPs in these candidate genes in an initial cohort of 54 children treated in pediatric oncology units, with replication in a second cohort of 112 children recruited through a national surveillance network for adverse drug reactions in Canada. We identified genetic variants in TPMT (rs12201199, P value = 0.00022, OR = 17.0, 95% CI 2.3-125.9) and COMT (rs9332377, P value = 0.00018, OR = 5.5, 95% CI 1.9-15.9) associated with cisplatin-induced hearing loss in children.

Therapeutic drug monitoring in patients with inflammatory bowel disease

PubMed Central

Yarur, Andres J; Abreu, Maria T; Deshpande, Amar R; Kerman, David H; Sussman, Daniel A

2014-01-01

Thiopurine analogs and anti-tumor necrosis factor (TNF) agents have dramatically changed the therapeutics of inflammatory bowel diseases (IBD), improving short and long-term outcomes. Unfortunately some patients do not respond to therapy and others lose response over time. The pharmacokinetic properties of these drugs are complex, with high inter-patient variability. Thiopurine analogs are metabolized through a series of pathways, which vary according to the patients’ pharmacogenetic profile. This profile largely determines the ratios of metabolites, which are in turn associated with likelihoods of clinical efficacy and/or toxicity. Understanding these mechanisms allows for manipulation of drug dose, aiming to reduce the development of toxicity while improving the efficacy of treatment. The efficacy of anti-TNF drugs is influenced by many pharmacodynamic variables. Several factors may alter drug clearance, including the concomitant use of immunomodulators (thiopurine analogs and methotrexate), systemic inflammation, the presence of anti-drug antibodies, and body mass. The treatment of IBD has evolved with the understanding of the pharmacologic profiles of immunomodulating and TNF-inhibiting medications, with good evidence for improvement in patient outcomes observed when measuring metabolic pathway indices. The role of routine measurement of metabolite/drug levels and antibodies warrants further prospective studies as we enter the era of personalized IBD care. PMID:24707130

Nature and position of functional group on thiopurine substrates influence activity of xanthine oxidase--enzymatic reaction pathways of 6-mercaptopurine and 2-mercaptopurine are different.

PubMed

Tamta, Hemlata; Kalra, Sukirti; Thilagavathi, Ramasamy; Chakraborti, Asit K; Mukhopadhyay, Anup K

2007-02-01

Xanthine oxidase-catalyzed hydroxylation reactions of the anticancer drug 6-mercaptopurine (6-MP) and its analog 2-mercaptopurine (2-MP) as well as 6-thioxanthine (6-TX) and 2-thioxanthine (2-TX) have been studied using UV-spectroscopy, high pressure liquid chromatography, photodiode array, and liquid chromatography-based mass spectral analysis. It is shown that 6-MP and 2-MP are oxidatively hydroxylated through different pathways. Enzymatic hydroxylation of 6-MP forms 6-thiouric acid in two steps involving 6-TX as the intermediate, whereas 2-MP is converted to 8-hydroxy-2-mercaptopurine as the expected end product in one step. Surprisingly, in contrast to the other thiopurines, enzymatic hydroxylation of 2-MP showed a unique hyperchromic effect at 264 nm as the reaction proceeded. However, when 2-TX is used as the substrate, it is hydroxylated to 2-thiouric acid. The enzymatic hydroxylation of 2-MP is considerably faster than that of 6-MP, while 6-TX and 2-TX show similar rates under identical reaction conditions. The reason why 2-MP is a better substrate than 6-MP and how the chemical nature and position of the functional groups present on the thiopurine substrates influence xanthine oxidase activity are discussed.

Therapeutic management of inflammatory bowel disease in real-life practice in the current era of anti-TNF agents: analysis of the French administrative health databases 2009-2014.

PubMed

Kirchgesner, J; Lemaitre, M; Rudnichi, A; Racine, A; Zureik, M; Carbonnel, F; Dray-Spira, R

2017-01-01

Management of inflammatory bowel disease (IBD) has evolved in the last decade. To assess IBD therapeutic management, including treatment withdrawal and early treatment use in the current era of anti-TNF agents (anti-TNFs). All patients affiliated to the French national health insurance diagnosed with IBD were included from 2009 to 2013 and followed up until 31 December 2014. Medication uses, treatment sequences after introduction of thiopurine or anti-TNF monotherapies or both (combination therapy), surgical procedures and hospitalisations were assessed. A total of 210 001 patients were diagnosed with IBD [Crohn's disease (CD), 100 112; ulcerative colitis (UC), 109 889]. Five years after diagnosis, cumulative probabilities of anti-TNF monotherapy and combination therapy exposures were 33.8% and 18.3% in CD patients and 12.9% and 7.4% in UC patients, respectively. Among incident patients who received thiopurines or anti-TNFs, the first treatment was thiopurine in 69.1% of CD and 78.2% of UC patients. Among patients treated with anti-TNFs, 45.2% and 54.5% of CD patients and 38.2% and 39.9% of UC patients started monotherapy and combination therapy within 3 months after diagnosis, respectively; 31.3% of CD and 27.1% of UC incident patients withdrew from thiopurine or anti-TNFs for more than 3 months after their first course of treatment. Five years after diagnosis, the cumulative risks of first intestinal resection in CD patients and colectomy in UC patients were 11.9% and 5.7%, respectively. Step-up approach remains the predominant strategy, while exposure to anti-TNFs is high. Surgery rates are low. Treatment withdrawal in IBD is more common than expected. © 2016 John Wiley & Sons Ltd.

Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?

PubMed

Vikingsson, Svante; Carlsson, Björn; Almer, Sven H C; Peterson, Curt

2009-06-01

Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.

Allopurinol might improve response to azathioprine and 6-mercaptopurine by correcting an unfavorable metabolite ratio.

PubMed

Gardiner, Sharon J; Gearry, Richard B; Burt, Michael J; Chalmers-Watson, Teresa; Chapman, Bruce A; Ross, Alison G; Stedman, Catherine A M; Huelsen, Alexander; Barclay, Murray L

2011-01-01

Allopurinol potentiates azathioprine and 6-mercaptopurine (6-MP) by increasing 6-thioguanine nucleotide (6-TGN) metabolite concentrations. The outcome might also be improved by adding allopurinol in individuals who preferentially produce 6-methylmercaptopurine nucleotides (6-MMPN), rather than 6-TGN. The aim of the present study was to investigate the effect of allopurinol on concentrations of 6-MMPN and 6-TGN in individuals with a high ratio of these metabolites (>20), which is indicative of a poor thiopurine response. Sixteen individuals were identified who were taking azathioprine or 6-MP, and were commenced on allopurinol to improve a high 6-MMPN:TGN ratio. Metabolite concentrations were compared before and after commencing allopurinol, and markers of disease control were compared. The addition of 100-300 mg allopurinol daily and thiopurine dose reduction (17-50% of the original dose) resulted in a reduction of the median (and range) 6-MMPN concentration, from 11,643 (3,365-27,832) to 221 (55-844) pmol/8×10(8) red blood cells (RBC; P=0.0005), increased 6-TGN from 162 (125-300) to 332 (135-923) pmol/8×10(8) RBC (P=0.0005), and reduced the 6-MMPN:6-TGN ratio from 63 (12-199) to 1 (0.1-4.5) (P=0.0005). There was a significant reduction in steroid dose requirements at 12 months (P=0.04) and trends for improvement in other markers of disease control. One patient developed red cell aplasia that resolved upon stopping azathioprine and allopurinol. In those with a high 6-MMPN:6-TGN ratio (>20), response to thiopurine treatment might be improved by the addition of allopurinol, together with a reduced thiopurine dose and close hematological monitoring. © 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

Risk of colectomy in patients with ulcerative colitis under thiopurine treatment.

PubMed

Cañas-Ventura, Alex; Márquez, Lucia; Ricart, Elena; Domènech, Eugeni; Gisbert, Javier P; García-Sanchez, Valle; Marín-Jiménez, Ignacio; Rodriguez-Moranta, Francisco; Gomollón, Fernando; Calvet, Xavier; Merino, Olga; Garcia-Planella, Esther; Vázquez-Romero, Narcis; Esteve, Maria; Iborra, Marisa; Gutiérrez, Ana; Vera, Maribel; Andreu, Montserrat

2014-10-01

Little is known about the risk factors of colectomy in patients with ulcerative colitis (UC) under thiopurine treatment. The aim of the study was to determine the prevalence and the predictive risk factors of colectomy in an extensive cohort of patients with UC treated with thiopurines in Spain. Among 5753 UC patients, we identified those diagnosed between 1980 and 2009 and treated with azathioprine or mercaptopurine (AZA/MP). We analyzed the age at diagnosis, familial history of IBD, extraintestinal manifestations (EIMs), disease extent, smoking status and treatment requirements (AZA/MP, cyclosporine (CsA) or anti-TNFα). Colectomies for dysplasia or cancer were excluded. Survival analysis and Cox proportional hazard regression were performed. Results were reported as hazard ratios (HR) with 95% CI. Among the 1334 cases included, 119 patients (8.9%) required colectomy after a median time of 26 months (IQR 12-42) after AZA/MP initiation. Independent predictors of colectomy were: Extensive UC (HR 1.7, 95% CI: 1.1-2.6), EIMs (HR 1.5, 95% CI: 1.0-2.4), need for antiTNFα (HR 2.3, 95% CI: 1.5-3.4) and need for CsA (HR 2.4, 95% CI: 1.6-3.7). Patients requiring early introduction of AZA/MP had an increased risk of colectomy with a HR of 4.9 (95% CI: 3.2-7.8) when AZA/MP started in the first 33 months after UC diagnosis. Nearly one-tenth of patients with UC under thiopurines require colectomy. Extensive UC, EIMs, need for CsA or anti-TNFα ever and an early need for AZA/MP treatment were associated with a higher risk of colectomy. These risk factors of colectomy could help to stratify risk in further controlled studies in UC. Copyright © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Excess primary intestinal lymphoproliferative disorders in patients with inflammatory bowel disease.

PubMed

Sokol, Harry; Beaugerie, Laurent; Maynadié, Marc; Laharie, David; Dupas, Jean-Louis; Flourié, Bernard; Lerebours, Eric; Peyrin-Biroulet, Laurent; Allez, Matthieu; Simon, Tabassome; Carrat, Fabrice; Brousse, Nicole

2012-11-01

It remains to be shown whether inflammatory bowel disease (IBD) is associated with an increased risk of primary intestinal lymphoproliferative disorders (PILD). We assessed this risk in the CESAME French nationwide prospective observational cohort. In all, 680 gastroenterologists enrolled 19,486 patients with IBD (Crohn's disease in 60.3%) from May 2004 to June 2005. Follow-up ended on 31 December 2007. Available biopsy samples and surgical specimens from patients with PILD (n = 14) were centralized for review. The reference incidence of PILD in the general population was obtained from the Côte d'Or registry and was used as a comparator to assess the standardized incidence ratio (SIR). The influence of thiopurine exposure was explored in a nested case-control study. In the CESAME population the crude incidence of PILD was 0.12/1000 patient-years, with a corresponding SIR of 17.51 (95% confidence interval [CI], 6.43-38.11; P < 0.0001). The risk was highest in patients exposed to thiopurines (SIR 49.52, 95% CI 13.49-126.8; P < 0.0001), while it did not reach statistical significance in patients naïve to thiopurines (SIR 4.83, 95% CI, 0.12-26.91; P = 0.37). The odds ratio associated with ongoing thiopurine exposure (vs. naïve) was 2.97 (95% CI, 0.30-infinity; P = 0.38). All 14 cases of PILD were non-Hodgkin's B-cell LD, 78.6% occurred in males, 85.7% arose in IBD lesions, and 45.5% were Epstein-Barr virus-positive. Eleven cases occurred in patients with Crohn's disease. Mean (SD) age at PILD diagnosis was 55.1 (5.6) years and the median time since IBD onset was 8.0 years (interquartile range, 3.0-15.8). Patients with IBD have an increased risk of developing PILD. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Inhibition of UDP-glucose dehydrogenase by 6-thiopurine and its oxidative metabolites: Possible mechanism for its interaction within the bilirubin excretion pathway and 6TP associated liver toxicity.

PubMed

Weeramange, Chamitha J; Binns, Cassie M; Chen, Chixiang; Rafferty, Ryan J

2018-03-20

6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a K i of 288 μM with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a K i of 7 μM. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with K i values of 54 and 14 μM, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities. Copyright © 2017 Elsevier B.V. All rights reserved.

Overall and Comparative Risk of Herpes Zoster With Pharmacotherapy for Inflammatory Bowel Diseases: A Nationwide Cohort Study.

PubMed

Khan, Nabeel; Patel, Dhruvan; Trivedi, Chinmay; Shah, Yash; Lichtenstein, Gary; Lewis, James; Yang, Yu-Xiao

2018-01-05

Patients with inflammatory bowel disease (IBD) might be at increased risk for herpes zoster infection. We sought to quantify the risk of herpes zoster in patients with IBD and evaluate the effects of IBD and IBD medications on the risk of herpes zoster. We conducted 2 retrospective studies of populations of Veterans, from January 2000 through June 2016. In study 1, we compared the incidence of herpes zoster among patients with IBD receiving 5-ASA alone vs matched patients without IBD. In study 2, we compared the incidence of herpes zoster among patients with IBD treated with only 5-ASA, with thiopurines, with antagonists of tumor necrosis factor (TNF), with a combination of thiopurines and TNF antagonists, and with vedolizumab. We used multivariable Cox regression to estimate the hazard ratios and 95% CIs for herpes zoster associated with IBD in study 1 and with different treatments in study 2. We also estimated the incidence rate of herpes zoster based on age and IBD medication subgroups. Compared to no IBD, ulcerative colitis (UC) and Crohn's disease (CD) were each associated with significantly increased risk of herpes zoster infection. In multivariable Cox regression (compared to no IBD), UC, CD, or IBD treated with 5-ASA treatment alone was associated with significantly increased risk of herpes zoster, with adjusted HRs (AHR) of 1.81 for UC (95% CI, 1.56-2.11), 1.56 for CD (95% CI, 1.28-1.91), and 1.72 for treated IBD (95% CI, 1.51-1.96). In multivariable Cox regression analysis, compared to exposure to 5-ASA alone, exposure to thiopurines (AHR, 1.47; 95% CI, 1.31-1.65) or a combination of thiopurines and TNF antagonists (AHR, 1.65; 95% CI, 1.22-2.23) was associated with increased risk of herpes zoster. However, exposure to TNF antagonists alone (AHR, 1.15; 95% CI, 0.96-1.38) was not associated with increased risk of herpes zoster. The incidence rates of herpes zoster in all age groups and all IBD medication subgroups were substantially higher than that in the oldest group of patients without IBD (older than 60 years). In 2 retrospective studies of Veteran populations, we associated IBD and treatment with thiopurines, alone or in combination with TNF antagonists, with increased risk of herpes zoster. With the approval of a new and potentially safer vaccine for herpes zoster, the effects of immunization of patients with IBD should be investigated. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Feline drug metabolism and disposition: pharmacokinetic evidence for species differences and molecular mechanisms

PubMed Central

2013-01-01

Synopsis Although it is widely appreciated that cats respond differently to certain drugs when compared with other companion animal species, the causes of these differences are poorly understood. This review critically evaluates published evidence for altered drug effects in cats, focusing on pharmacokinetic differences between cats, dogs and humans, and the molecular mechanisms underlying these differences. Pharmacokinetic studies indicate that acetaminophen, propofol, carprofen, and acetylsalicylic acid (aspirin) are cleared significantly more slowly in cats versus dogs and humans. All of these drugs are metabolized by conjugation. Cats lack the major phenol UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6 and UGT1A9, that glucuronidate acetaminophen and propofol. Deficient glucuronidation may also explain slower carprofen clearance, although there is no direct evidence for this. However, poor aspirin clearance in cats appears to be mainly a consequence of slower glycine conjugation. Cats are also deficient in several other conjugation enzymes, including N-acetyltransferase (NAT) 2 and thiopurine methyltransferase (TMPT). NAT2 deficiency may be the reason cats are more prone to developing methemoglobinemia rather than hepatotoxicity from acetaminophen. TMPT deficiency may predispose cats to azathioprine toxicity. No evidence was found for slower elimination of drugs cleared by oxidation or unchanged into urine or bile. Piroxicam, an oxidized drug, was cleared much more rapidly in cats than humans and dogs, although the mechanism for this difference is unclear. More work is needed to better understand drug metabolism and disposition differences in cats, thereby enabling more rational prescribing of existing medications, and the development of safer drugs for this species. PMID:23890237

[Long-term therapy of idiopathic inflammatory bowel disease].

PubMed

Lukáš, K; Dastych, M; Novotný, A; Prokopová, L; Zbořil, V

2012-01-01

Crohns disease and ulcerative colitis are chronic inflammatory diseases of the gastrointestinal tract. Both can be treated with medications that induce and maintain remission. The choice of medication is influenced by the balance between drug potency and potential side-effects, previous response to treatment, and the presence of extraintestinal manifestations or complications. After remission has been achieved, the goal of treatment is to maintain the symptom-free status. 5-aminosalicylic acid derivatives have efficacy for maintenance of remission in patients with distal disease. Thiopurines are recommended for the long-term therapy. For the patients who do not have a response to immunosuppressive therapy or cannot tolerate it, anti-TNF-α agents are gradually being adopted. Effective in the remission maintenance are thiopurines, infliximab and adalimumab.

Liquid chromatography-mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients.

PubMed

Coulthard, Sally A; Berry, Phil; McGarrity, Sarah; Ansari, Azhar; Redfern, Christopher P F

2016-08-15

Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20-1360mol dTG/10(6)mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Tailoring density and optical and thermal behavior of gold surfaces and nanoparticles exploiting aromatic dithiols.

PubMed

Bruno, Giovanni; Babudri, Francesco; Operamolla, Alessandra; Bianco, Giuseppe V; Losurdo, Maria; Giangregorio, Maria M; Hassan Omar, Omar; Mavelli, Fabio; Farinola, Gianluca M; Capezzuto, Pio; Naso, Francesco

2010-06-01

Self-assembled monolayers (SAMs) derived of 4-methoxy-terphenyl-3'',5''-dimethanethiol (TPDMT) and 4-methoxyterphenyl-4''-methanethiol (TPMT) have been prepared by chemisorption from solution onto gold thin films and nanoparticles. The SAMs have been characterized by spectroscopic ellipsometry, Raman spectroscopy and atomic force microscopy to determine their optical properties, namely the refractive index and extinction coefficient, in an extended spectral range of 0.75-6.5 eV. From the analysis of the optical data, information on SAMs structural organization has been inferred. Comparison of SAMs generated from the above aromatic thiols to well-known SAMs generated from the alkanethiol dodecanethiol revealed that the former aromatic SAMs are densely packed and highly vertically oriented, with a slightly higher packing density and a absence of molecular inclination in TPMT/Au. The thermal behavior of SAMs has also been monitored using ellipsometry in the temperature range 25-500 degrees C. Gold nanoparticles functionalized by the same aromatic thiols have also been discussed for surface enhanced Raman spectroscopy applications. This study represents a step forward tailoring the optical and thermal behavior of surfaces as well as nanoparticles.

Synthesis and antitumor activity of seleno- and thio-purines complexed with cis-diamminoplatinum (II).

PubMed

Maeda, M; Abiko, N; Sasaki, T

1982-02-01

cis-Diamminoplatinum (II) complexes with selenoguanine, thioguanine, 6-thioxanthine, or 6-mercaptopurine were synthesized by the reaction of stoichiometric amounts of selenopurine or thiopurine with aquated cis-dichlorodimmineplatinum (II) in slightly acidic medium, and their antitumor activity was studied against L1210 cells in mice. These compounds exhibited a medium antitumor activity with very low toxicity. The antitumor activity was dependent on the nature of the purine ligand. These complexes were very stable in various aqueous solvents at 37 degrees C for 10 d but not in the presence of mouse serum. The mechanism of the action effected by the complex is not clear. However, the slow release of an antitumor active purine from the complex, SeG-Pt (NH3)2, was observed.

Crohn disease - discharge

MedlinePlus

... Inadomi JM, Hanauer SB. American Gastroenterological Association Institute technical review on the use of thiopurines, methotrexate, and ... Duplication for commercial use must be authorized in writing by ADAM Health Solutions. About MedlinePlus Site Map ...

Ulcerative colitis - discharge

MedlinePlus

... Inadomi JM, Hanauer SB. American Gastroenterological Association Institute technical review on the use of thiopurines, methotrexate, and ... Duplication for commercial use must be authorized in writing by ADAM Health Solutions. About MedlinePlus Site Map ...

Concomitant Use of Immunomodulators Affects the Durability of Infliximab Therapy in Children With Crohn's Disease.

PubMed

Grossi, Victoria; Lerer, Trudy; Griffiths, Anne; LeLeiko, Neal; Cabrera, Jose; Otley, Anthony; Rick, James; Mack, David; Bousvaros, Athos; Rosh, Joel; Grossman, Andrew; Saeed, Shehzaad; Kay, Marsha; Boyle, Brendan; Oliva-Hemker, Maria; Keljo, David; Pfefferkorn, Marian; Faubion, William; Kappelman, Michael D; Sudel, Boris; Markowitz, James; Hyams, Jeffrey S

2015-10-01

It is important to determine the effects of immunomodulators on the ability of children to remain on infliximab therapy for Crohn's disease (durability of therapy), given the potential benefits and risks of concomitant therapy-especially with thiopurines in male patients. We investigated how immunomodulatory treatment affects the durability of infliximab therapy. We collected data from the Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry, from January 2002 through August 2014, on 502 children with Crohn's disease who participated in a prospective multicenter study. Data were collected from patients who received at least a 3-dose induction regimen of infliximab, and their concomitant use of immunomodulators: no thiopurine or methotrexate treatment, treatment for 6 months or less during infliximab therapy, or treatment for more than 6 months during infliximab therapy. The probabilities (± standard error) that children remained on infliximab therapy for 1 year, 3 years, and 5 years after the treatment began were 0.84 ± 0.02, 0.69 ± 0.03, and 0.60 ± 0.03, respectively. Age, sex, and disease extent or location did not affect the durability of infliximab therapy. Greater length of concomitant use of immunomodulators was associated with increased time of infliximab therapy. The probability that patients with more than 6 months of immunomodulator use remained on infliximab therapy for 5 years was 0.70 ± 0.04, compared with 0.48 ± 0.08 for patients who did not receive immunomodulators and 0.55 ± 0.06 for patients who received immunomodulators for 6 months or less (P < .001). In boys who received immunomodulators for 6 months or more after starting infliximab, the overall durability of infliximab therapy was greater among patients receiving methotrexate than thiopurine (P < .01); the probabilities that they remained on infliximab therapy for 5 years were 0.97 ± 0.03 vs 0.58 ± 0.08, respectively. In children with Crohn's disease, concomitant treatment with an immunomodulator for more than 6 months after starting infliximab therapy increases the chances that patients will remain on infliximab. In boys, methotrexate appears to increase the durability of infliximab therapy compared with thiopurine. Copyright © 2015. Published by Elsevier Inc.

Synthesis of Lysine Methyltransferase Inhibitors

NASA Astrophysics Data System (ADS)

Ye, Tao; Hui, Chunngai

2015-07-01

Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

Physician Perspectives on Unresolved Issues in the Management of Ulcerative Colitis: The UC Horizons Project.

PubMed

Gisbert, Javier P; Barreiro-de Acosta, Manuel; Esteve, María; García-Sánchez, Valle; Gomollón, Fernando; Guardiola, Jordi; Hinojosa, Joaquin; Martín Arranz, Maria-Dolores; Minguez, Miguel; Taxonera, Carlos; Vera, Isabel

2016-03-01

There is still uncertainty about what constitutes the best therapeutic practice in ulcerative colitis (UC). The purpose of the "UC Horizons Project" was to raise a series of questions regarding the management of UC to provide responses based on the best scientific evidence available. The 11 members of the scientific committee prepared draft answers to the 10 questions from available evidence after a literature search. A total of 48 Spanish gastroenterology specialists nationwide participated in the project. The national meeting discussed the 10 issues in working groups and reached consensus regarding the recommendations by anonymous, interactive vote following the Delphi methodology. Final answers were developed, based on evidence and clinical experience of the participants. All the recommendations achieved a high level of agreement in the plenary vote, although the quality of the evidence was markedly heterogeneous. The lowest percentage of agreement corresponded to the questions with the weakest level of evidence, highlighting the necessity of conducting further studies in these areas. The recommendations focused on (1) aminosalicylates therapy (regarding dose and appropriateness of coadministration with thiopurines), (2) corticosteroid therapy (regarding dose and route of administration), (3) thiopurine treatment (regarding indications and possibility of withdrawal), (4) anti-tumor necrosis factor therapy (regarding appropriateness of combination with thiopurines, intensification, or discontinuation of treatment), and (5) colorectal cancer (regarding risk and time trends). The UC Horizons Project raised a series of eminently practical questions about the management of UC and provided responses based on the best scientific evidence available.

The degree of myelosuppression during maintenance therapy of adolescents with B-lineage intermediate risk acute lymphoblastic leukemia predicts risk of relapse.

PubMed

Schmiegelow, K; Heyman, M; Gustafsson, G; Lausen, B; Wesenberg, F; Kristinsson, J; Vettenranta, K; Schroeder, H; Forestier, E; Rosthoej, S

2010-04-01

Drug doses, blood levels of drug metabolites and myelotoxicity during 6-mercaptopurine/methotrexate (MTX) maintenance therapy were registered for 59 adolescents (>or=10 years) and 176 non-adolescents (<10 years) with B-cell precursor acute lymphoblastic leukemia (ALL) and a white blood cell count (WBC) <50 x 10(9)/l at diagnosis. Event-free survival was lower for adolescents than non-adolescents (pEFS(12y):0.71 vs 0.83, P=0.04). For adolescents staying in remission, the mean WBC during maintenance therapy (mWBC) was related to age (r(S)=0.36, P=0.02), which became nonsignificant for those who relapsed (r(S)=0.05, P=0.9). The best-fit multivariate Cox regression model to predict risk of relapse included mWBC and thiopurine methyltransferase activity, which methylates mercaptopurine and reduces the intracellular availability of cytotoxic 6-thioguanine nucleotides (coefficient: 0.11, P=0.02). The correlation of mWBC to the risk of relapse was more pronounced for adolescents (coefficient=0.65, P=0.003) than for non-adolescents (coefficient=0.42, P=0.04). Adolescents had higher mean neutrophil counts (P=0.002) than non-adolescents, but received nonsignificantly lower mercaptopurine and MTX doses during maintenance therapy. Red blood cell MTX levels were significantly related to the dose of MTX among adolescents who stayed in remission (r(S)=0.38, P=0.02), which was not the case for those who developed a relapse (r(S)=0.15, P=0.60). Thus, compliance to maintenance therapy may influence the risk of relapse for adolescents with ALL.

Pharmacoeconomic evaluations of pharmacogenetic and genomic screening programmes: a systematic review on content and adherence to guidelines.

PubMed

Vegter, Stefan; Boersma, Cornelis; Rozenbaum, Mark; Wilffert, Bob; Navis, Gerjan; Postma, Maarten J

2008-01-01

The fields of pharmacogenetics and pharmacogenomics have become important practical tools to progress goals in medical and pharmaceutical research and development. As more screening tests are being developed, with some already used in clinical practice, consideration of cost-effectiveness implications is important. A systematic review was performed on the content of and adherence to pharmacoeconomic guidelines of recent pharmacoeconomic analyses performed in the field of pharmacogenetics and pharmacogenomics. Economic analyses of screening strategies for genetic variations, which were evidence-based and assumed to be associated with drug efficacy or safety, were included in the review. The 20 papers included cover a variety of healthcare issues, including screening tests on several cytochrome P450 (CYP) enzyme genes, thiopurine S-methyltransferase (TMPT) and angiotensin-converting enzyme (ACE) insertion deletion (ACE I/D) polymorphisms. Most economic analyses reported that genetic screening was cost effective and often even clearly dominated existing non-screening strategies. However, we found a lack of standardization regarding aspects such as the perspective of the analysis, factors included in the sensitivity analysis and the applied discount rates. In particular, an important limitation of several studies related to the failure to provide a sufficient evidence-based rationale for an association between genotype and phenotype. Future economic analyses should be conducted utilizing correct methods, with adherence to guidelines and including extensive sensitivity analyses. Most importantly, genetic screening strategies should be based on good evidence-based rationales. For these goals, we provide a list of recommendations for good pharmacoeconomic practice deemed useful in the fields of pharmacogenetics and pharmacogenomics, regardless of country and origin of the economic analysis.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

PubMed

Matsuoka, Taeko; Kawai, Koji; Ando, Satoshi; Sugita, Shintaro; Kandori, Shuya; Kojima, Takahiro; Miyazaki, Jun; Nishiyama, Hiroyuki

2016-05-01

DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

PubMed

Gou, Qing; He, ShuJiao; Zhou, ZeJian

2017-02-01

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

PubMed

Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

2014-02-01

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine.

PubMed Central

Stefl, R; Spacková, N; Berger, I; Koca, J; Sponer, J

2001-01-01

The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses. PMID:11159416

Clinical course of ulcerative colitis patients who develop acute pancreatitis

PubMed Central

Kim, Jong Wook; Hwang, Sung Wook; Park, Sang Hyoung; Song, Tae Jun; Kim, Myung-Hwan; Lee, Ho-Su; Ye, Byong Duk; Yang, Dong-Hoon; Kim, Kyung-Jo; Byeon, Jeong-Sik; Myung, Seung-Jae; Yang, Suk-Kyun

2017-01-01

AIM To investigate the clinical course of ulcerative colitis (UC) patients who develop acute pancreatitis. METHODS We analyzed 3307 UC patients from the inflammatory bowel disease registry at Asan Medical Center from June 1989 to May 2015. The clinical course of UC patients who developed acute pancreatitis was compared with that of non-pancreatitis UC patients. RESULTS Among 51 patients who developed acute pancreatitis, 13 (0.40%) had autoimmune, 10 (0.30%) had aminosalicylate-induced, and 13 (1.73%) had thiopurine-induced pancreatitis. All 13 patients with autoimmune pancreatitis (AIP) had type 2 AIP. Two (15.4%) patients had pre-existing AIP, and three (23.1%) patients developed AIP and UC simultaneously. Compared to non-pancreatitis patients, AIP patients had UC diagnosed at a significantly younger age (median, 22.9 years vs 36.4 years; P = 0.001). AIP and aminosalicylate-induced pancreatitis patients had more extensive UC compared to non-pancreatitis patients. All patients with pancreatitis recovered uneventfully, and there were no recurrences. Biologics were used more frequently in aminosalicylate- and thiopurine-induced pancreatitis patients compared to non-pancreatitis patients [adjusted OR (95%CI), 5.16 (1.42-18.67) and 6.90 (1.83-25.98), respectively]. Biologic utilization rate was similar among AIP and non-pancreatitis patients [OR (95%CI), 0.84 (0.11-6.66)]. Colectomy rates for autoimmune, aminosalicylate-induced, and thiopurine-induced pancreatitis, and for non-pancreatitis patients were 15.4% (2/13), 20% (2/10), 15.4% (2/13), and 7.3% (239/3256), respectively; the rates were not significantly different after adjusting for baseline disease extent. CONCLUSION Pancreatitis patients show a non-significant increase in colectomy, after adjusting for baseline disease extent. PMID:28596686

Clinical course of ulcerative colitis patients who develop acute pancreatitis.

PubMed

Kim, Jong Wook; Hwang, Sung Wook; Park, Sang Hyoung; Song, Tae Jun; Kim, Myung-Hwan; Lee, Ho-Su; Ye, Byong Duk; Yang, Dong-Hoon; Kim, Kyung-Jo; Byeon, Jeong-Sik; Myung, Seung-Jae; Yang, Suk-Kyun

2017-05-21

To investigate the clinical course of ulcerative colitis (UC) patients who develop acute pancreatitis. We analyzed 3307 UC patients from the inflammatory bowel disease registry at Asan Medical Center from June 1989 to May 2015. The clinical course of UC patients who developed acute pancreatitis was compared with that of non-pancreatitis UC patients. Among 51 patients who developed acute pancreatitis, 13 (0.40%) had autoimmune, 10 (0.30%) had aminosalicylate-induced, and 13 (1.73%) had thiopurine-induced pancreatitis. All 13 patients with autoimmune pancreatitis (AIP) had type 2 AIP. Two (15.4%) patients had pre-existing AIP, and three (23.1%) patients developed AIP and UC simultaneously. Compared to non-pancreatitis patients, AIP patients had UC diagnosed at a significantly younger age (median, 22.9 years vs 36.4 years; P = 0.001). AIP and aminosalicylate-induced pancreatitis patients had more extensive UC compared to non-pancreatitis patients. All patients with pancreatitis recovered uneventfully, and there were no recurrences. Biologics were used more frequently in aminosalicylate- and thiopurine-induced pancreatitis patients compared to non-pancreatitis patients [adjusted OR (95%CI), 5.16 (1.42-18.67) and 6.90 (1.83-25.98), respectively]. Biologic utilization rate was similar among AIP and non-pancreatitis patients [OR (95%CI), 0.84 (0.11-6.66)]. Colectomy rates for autoimmune, aminosalicylate-induced, and thiopurine-induced pancreatitis, and for non-pancreatitis patients were 15.4% (2/13), 20% (2/10), 15.4% (2/13), and 7.3% (239/3256), respectively; the rates were not significantly different after adjusting for baseline disease extent. Pancreatitis patients show a non-significant increase in colectomy, after adjusting for baseline disease extent.

Long-term Safety and Efficacy of Low-dose Azathioprine and Allopurinol Cotherapy in Inflammatory Bowel Disease: A Large Observational Study.

PubMed

Pavlidis, Polychronis; Stamoulos, Panagiotis; Abdulrehman, Answar; Kerr, Patrick; Bull, Claire; Duley, John; Ansari, Azhar

2016-07-01

Low-dose azathioprine with allopurinol (LDAA) has been proposed as a potent therapy in inflammatory bowel disease (IBD) with the benefit of overcoming side effects regularly associated with thiopurine monotherapy and poor responses. Concerns regarding safety remain, while a layer of complexity has been added by the trend toward treatment directed by red cell thioguanine nucleotide (TGN) profiling. We report on the clinical efficacy and safety of LDAA use in IBD undirected by metabolite profiling. Observational study of clinical practice from a single IBD center. Patient outcomes were defined clinically based on established activity scores and corticosteroid withdrawal. Red cell TGN was monitored only for suspected nonadherence. Overall, 113/164 (69%) patients with Crohn's disease and 83/136 (61%) patients with ulcerative/unclassified colitis had a clinical response by the end of follow-up (median 19 months), while 85 (52%) patients with Crohn's disease and 74 (54%) patients with ulcerative/unclassified colitis were in clinical remission. Clinical response was seen in 45/57 (79%) patients with Crohn's disease and 34/53 (64%) patients with ulcerative/unclassified colitis who were thiopurine naive, had active IBD, and received LDAA as the first line immunomodulator, while in 35 (61%) and 28 (53%), respectively, remission was achieved. LDAA was stopped in 20/300 (7%) patients because of side effects, all of which resolved on drug cessation. This is the largest cohort supporting the favorable safety profile and high efficacy of LDAA in IBD. It presents 2 advances in therapy: prescribing LDAA for thiopurine-naive patients, and bypassing TGN monitoring in favor of clinical monitoring (blood counts, etc.), which will make it more accessible for clinics without access to TGN assays.

Determination of urinary 6-mercaptopurine and three of its metabolites by HPLC-UV coupled with the iodine-azide reaction.

PubMed

Zakrzewski, Robert; Borowczyk, Kamila; Łuczak, Adam; Młynarski, Wojciech; Trelińska, Joanna

2013-04-01

The presented method is able to determine 6-mercaptopurine (6-MP), 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside in urine, and is thereby dedicated to control of thiopurine therapy of children with acute lymphoblastic leukemia. Good separation of the mentioned compounds was achieved on a C18 stationary phase with a sodium azide and sodium heptane sulfonate solution, acetonitrile and water at ratio of 50:1:49 (v/v/v). Coefficient of regression is >0.99 for all linearity ranges. LOD and LOQ are 0.3, 0.4, 0.3, 0.8 and 0.4, 0.6, 0.5 and 0.9 nmol/ml of urine for 6-MP, 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside, respectively. Intra- and inter-day recovery and RSD are close to 100% and less than 10%, respectively, for all investigated thiopurines. The elaborated method was successfully applied for detection and quantitation of 6-MP and its selected metabolites in patients' urine samples.

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants.

PubMed

Grebenok, R J; Galbraith, D W; Penna, D D

1997-08-01

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GALA regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.

Risk of Nonmelanoma Skin Cancer Associated With the Use of Immunosuppressant and Biologic Agents in Patients With a History of Autoimmune Disease and Nonmelanoma Skin Cancer.

PubMed

Scott, Frank I; Mamtani, Ronac; Brensinger, Colleen M; Haynes, Kevin; Chiesa-Fuxench, Zelma C; Zhang, Jie; Chen, Lang; Xie, Fenglong; Yun, Huifeng; Osterman, Mark T; Beukelman, Timothy; Margolis, David J; Curtis, Jeffrey R; Lewis, James D

2016-02-01

Immune dysfunction underlies the pathogenesis of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Immunosuppressive therapy is the standard of care for these diseases. Both immune dysfunction and therapy-related immunosuppression can inhibit cancer-related immune surveillance in this population. Drug-induced immunosuppression is a risk factor for nonmelanoma skin cancer (NMSC), particularly squamous cell tumors. For patients with a history of NMSC, data are limited on the effect of these drugs on the risk of additional NMSCs. To determine the relative hazard of a second NMSC in patients with RA or IBD who use methotrexate, anti-tumor necrosis factor (anti-TNF) therapy, or thiopurines after an initial NMSC. In this retrospective cohort study, we studied 9460 individuals with RA or IBD enrolled in Medicare from January 1, 2006, through December 31, 2012. Exposure to methotrexate, thiopurines, anti-TNFs, sulfasalazine, hydroxychloroquine, abatacept, or rituximab after the incident NMSC surgery. A second NMSC occurring 1 year or more after the incident NMSC using Cox proportional hazards regression models. Among 9460 individuals (6841 with RA and 2788 with IBD), the incidence rate of a second NMSC per 1000 person-years was 58.2 (95% CI, 54.5-62.1) and 58.9 (95% CI, 53.2-65.2) in patients with RA and IBD, respectively. Among patients with RA, methotrexate used in conjunction with other medications was associated with an increased risk of a second NMSC (hazard ratio [HR], 1.60; 95% CI, 1.08-2.37). Adjusted for other medications, the risk of NMSC increased with 1 year or more of methotrexate use (HR, 1.24; 95% CI, 1.04-1.48). Compared with methotrexate alone, the addition of anti-TNF drugs was significantly associated with risk of NMSC (HR, 1.49; 95% CI, 1.03-2.16). Abatacept and rituximab were not associated with increased NMSC risk. The nonsignificant HRs for 1 year or more of thiopurine and anti-TNF use for IBD were 1.49 (95% CI, 0.98-2.27) and 1.36 (95% CI, 0.76-2.44), respectively. Methotrexate use is associated with an increased risk of a second NMSC. Anti-TNF use may increase the risk of a second NMSC when used with methotrexate for RA. Further long-term studies are required before one can conclude that thiopurine and/or anti-TNF do not increase the risk of a second NMSC in patients with IBD.

A Panoptic Uncovering of the Dynamical Evolution of the Zika Virus NS5 Methyltransferase Binding Site Loops- Zeroing in on the Molecular Landscape.

PubMed

Devnarain, Nikita; Soliman, Mahmoud E S

2018-06-20

The global threat of the Zika virus to humanity is real. Innovative and potent anti-Zika virus drugs are still at large, due to the lack of anti-Zika virus drugs that have passed phase 1 trials. Experimental research has revealed novel inhibitors of Zika virus NS5 methyltransferase enzyme. This study has taken a step further to provide insight into the molecular dynamics of Zika virus and inhibitor binding, which have not been established experimentally. Movements of the methyltransferase binding site loops have a large role to play in the methylation of the viral mRNA cap, which is essential for Zika virus replication. Here we pinpoint the binding interactions between each potential inhibitor and the methyltransferase, residues that are responsible for binding, as well as which inhibitor-bound complex renders the methyltransferase more stable. We also highlight the conformational changes that occur within the methyltransferase to accommodate binding of inhibitors and consequences of those changes upon the RNA- and cap-binding sites in the methyltransferase. This research will improve the understanding of the Zika virus NS5 methyltransferase enzyme, and will be beneficial in driving the development of anti-Zika virus drugs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

PubMed

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

PubMed

Pillinger, M H; Volker, C; Stock, J B; Weissmann, G; Philips, M R

1994-01-14

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

PubMed

Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

2000-12-07

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea*S⃞

PubMed Central

Ferguson, Tsuneo; Soares, Jitesh A.; Lienard, Tanja; Gottschalk, Gerhard; Krzycki, Joseph A.

2009-01-01

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function. PMID:19043046

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

PubMed Central

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

2011-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-04-15

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed Central

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-01-01

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation. PMID:14705965

Challenges in profiling and lead optimization of drug discovery for methyltransferases.

PubMed

Horiuchi, Kurumi Y

2015-11-01

The importance of epigenetics in the initiation and progression of disease has attracted many investigators to incorporate this novel and exciting field in drug development. Protein methyltransferases are one of the target classes which have gained attention as potential therapeutic targets after promising results of inhibitors for EZH2 and DOT1L in clinical trials. There are many technologies developed in order to find small molecule inhibitors for protein methyltransferases. However, in contrast to high throughput screening, profiling against different methyltransferases is challenging since each enzyme has a different substrate preference so that it is hard to profile in one assay format. Here, different technologies for methyltransferase assays will be overviewed, and the advantages and disadvantages of each will be discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of brevetoxin exposure on the immune system of loggerhead sea turtles.

PubMed

Walsh, Catherine J; Leggett, Stephanie R; Carter, Barbara J; Colle, Clarence

2010-05-10

Blooms of the toxic dinoflagellate, Karenia brevis, occur almost annually off the Florida coast. These blooms, commonly called "red tides", produce a group of neurotoxins collectively termed brevetoxins. Many species of sealife, including sea turtles, are severely impacted by brevetoxin exposure. Effects of brevetoxins on immune cells were investigated in rescued loggerhead sea turtles, Caretta caretta, as well as through in vitro experiments using peripheral blood leukocytes (PBL) collected from captive sea turtles. In rescued animals, plasma brevetoxin concentrations were measured using a competitive ELISA. Plasma lysozyme activity was measured using a turbidity assay. Lysozyme activity correlated positively with plasma brevetoxin concentrations. Differential expression of genes affected by brevetoxin exposure was determined using two separate suppression subtractive hybridization experiments. In one experiment, genes from PBL collected from sea turtles rescued from red tide toxin exposure were compared to genes from PBL collected from healthy captive loggerhead sea turtles. In the second experiment, PBL from healthy captive loggerhead sea turtles were exposed to brevetoxin (500 ng PbTx-2/ml) in vitro for 18 h and compared to unexposed PBL. Results from the subtraction hybridization experiment conducted with red tide rescued sea turtle PBL indicated that genes involved in oxidative stress or xenobiotic metabolism were up-regulated. Using quantitative real-time PCR, a greater than 2-fold increase in superoxide dismutase and thioredoxin and greater than 10-fold increase in expression of thiopurine S-methyltransferase were observed. Results from the in vitro subtraction hybridization experiment indicated that genes coding for cytochrome c oxidases were the major up-regulated genes. Using quantitative real-time PCR, a greater than 8-fold increase in expression of beta-tubulin and greater than 3-fold increase in expression of ubiquinol were observed. Brevetoxin exposure may have significant implications for immune function in loggerhead sea turtles. 2009 Elsevier B.V. All rights reserved.

Characterization of an O-Demethylase of Desulfitobacterium hafniense DCB-2

PubMed Central

Studenik, Sandra; Vogel, Michaela

2012-01-01

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (∼70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein. PMID:22522902

Hemophagocytic lymphohistiocytosis caused by primary Epstein-Barr virus in patient with Crohn's disease.

PubMed

Virdis, Francesco; Tacci, Sara; Messina, Federico; Varcada, Massimo

2013-11-27

We present a case of a 19-year-old man with a 6-year history of Crohn's disease (CD), previously treated with 6-mercaptopurine, who was admitted to our department for Epstein-Barr virus (EBV) infection and subsequently developed a hemophagocytic lymphohistiocytosis (HLH). HLH is a rare disease which causes phagocytosis of all bone marrow derived cells. It can be a primary form as a autosomic recessive disease, or a secondary form associated with a variety of infections; EBV is the most common, the one with poorer prognosis. The incidence of lymphoproliferative disorders was increased in patients with inflammatory bowel disease (IBD) treated with thiopurines. Specific EBV-related clinical and virological management should be considered when treating a patient with IBD with immunosuppressive therapy. Moreover EBV infection in immunosuppressed patient can occur with more aggressive forms such as encephalitis and diffuse large B cell lymphoma. Our case confirms what is described in the literature; patients with IBD, particularly patients with CD receiving thiopurine therapy, who present 5 d of fever and cervical lymphadenopathy or previous evidence of lymphopenia should be screened for HLH.

A simplified characterization of S-adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting.

PubMed

Burgos, Emmanuel S; Walters, Ryan O; Huffman, Derek M; Shechter, David

2017-09-01

Methyltransferases use S -adenosyl-l-methionine (SAM) to deposit methyl marks. Many of these epigenetic 'writers' are associated with gene regulation. As cancer etiology is highly correlated with misregulated methylation patterns, methyltransferases are emerging therapeutic targets. Successful assignment of methyltransferases' roles within intricate biological networks relies on (1) the access to enzyme mechanistic insights and (2) the efficient screening of chemical probes against these targets. To characterize methyltransferases in vitro and in vivo , we report a highly-sensitive one-step deaminase-linked continuous assay where the S -adenosyl-l-homocysteine (SAH) enzyme-product is rapidly and quantitatively catabolized to S -inosyl-l-homocysteine (SIH). To highlight the broad capabilities of this assay, we established enzymatic characteristics of two protein arginine methyltransferases (PRMT5 and PRMT7), a histone-lysine N -methyltransferase (DIM-5) and a sarcosine/dimethylglycine N -methyltransferase (SDMT). Since the coupling deaminase TM0936 displays robust activity over a broad pH-range we determined the pH dependence of SDMT reaction rates. TM0936 reactions are monitored at 263 nm, so a drawback may arise when methyl acceptor substrates absorb within this UV-range. To overcome this limitation, we used an isosteric fluorescent SAM-analog: S -8-aza-adenosyl-l-methionine. Most enzymes tolerated this probe and sustained methyltransfers were efficiently monitored through loss of fluorescence at 360 nm. Unlike discontinuous radioactive- and antibody-based assays, our assay provides a simple, versatile and affordable approach towards the characterization of methyltransferases. Supported by three logs of linear dynamic range, the 1-Step EZ-MTase can detect methylation rates as low as 2 μM h -1 , thus making it possible to quantify low nanomolar concentrations of glycine N -methyltransferase within crude biological samples. With Z '-factors above 0.75, this assay is well suited to high-throughput screening and may promote the identification of novel therapeutics.

Influence of 5-aminosalicylic acid on 6-thioguanosine phosphate metabolite levels: a prospective study in patients under steady thiopurine therapy

PubMed Central

de Graaf, P; de Boer, NKH; Wong, DR; Karner, S; Jharap, B; Hooymans, PM; Veldkamp, AI; Mulder, CJJ; van Bodegraven, AA; Schwab, M

2010-01-01

Background and purpose: 5-aminosalicylate (5-ASA) raises levels of 6-thioguanine nucleotides (6-TGN), the active metabolites of thiopurines such as azathioprine (AZA). Changes in levels of each individual TGN – 6-thioguanosine mono-, di- and triphosphate (6-TGMP, 6-TGDP, 6-TGTP) – and of 6-methylmercaptopurine ribonucleotides (6-MMPR) after 5-ASA are not known. Experimental approach: Effects of increasing 5-ASA doses on AZA metabolites were investigated prospectively in 22 patients with inflammatory bowel disease in 4-week study periods. Patients started with 2 g 5-ASA daily, and then were increased to 4 g daily and followed by a washout period. Thiopurine doses remained unchanged throughout the entire study. Levels of 6-TGMP, 6-TGDP, 6-TGTP and 6-MMPR as well as of 5-ASA and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) were determined each study period. Key results: Median baseline levels in 17 patients of 6-TGDP, 6-TGTP and 6-MMPR were 52, 319 and 1676 pmol per 8 × 108 red blood cells respectively. After co-administration of 2 g 5-ASA daily, median 6-TGDP and 6-TGTP levels increased but median 6-MMPR levels were unchanged. Increasing 5-ASA to 4 g daily did not affect median 6-TGDP and 6-TGTP levels, but median 6-MMPR levels decreased. After discontinuation of 5-ASA, both 6-TGDP and 6-TGTP levels decreased and median 6-MMPR levels increased. The 6-TGTP/(6-TGDP+6-TGTP)-ratio did not change during the study, but 6-MMPR/6-TGN ratios decreased. Conclusions and implications: Individual 6-TGN metabolites increased after addition of 5-ASA, but 6-MMPR-levels and the 6-MMPR/6-TGN ratios decreased. Further studies are needed to decide whether this pharmacokinetic interaction would result in improvement of efficacy and/or increased risk of toxicity of AZA. PMID:20590602

Thiopurine Therapy Reduces the Incidence of Colorectal Neoplasia in Patients with Ulcerative Colitis. Data from the ENEIDA Registry.

PubMed

Gordillo, Jordi; Cabré, Eduard; Garcia-Planella, Esther; Ricart, Elena; Ber-Nieto, Yolanda; Márquez, Lucía; Rodríguez-Moranta, Francisco; Ponferrada, Ángel; Vera, Isabel; Gisbert, Javier P; Barrio, Jesús; Esteve, Maria; Merino, Olga; Muñoz, Fernando; Domènech, Eugeni

2015-12-01

Patients with ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC), but recent studies suggest a lower risk than previously reported. The aim was to evaluate the incidence of dysplasia, CRC and related risk factors in UC patients from a Spanish nationwide database. All UC patients were identified and retrospectively reviewed. Clinical-epidemiological data and the finding of dysplasia and/or CRC were collected. A total of 831 UC patients were included. Twenty-six cases of CRC in 26 patients and 29 cases of high-grade dysplasia (HGD) in 24 patients were found, accounting for 55 diagnoses of advanced neoplasia (AN = CRC and/or HGD) in 45 patients (33% of them within the first 8 years after UC diagnosis). The cumulative risk of AN was 2, 5.3 and 14.7% at 10, 20 and 30 years, respectively. Concomitant primary sclerosing cholangitis (odds ratio [OR] 10.90; 95% confidence interval [CI] 3.75-31.76, p < 0.001), extensive UC (OR 2.10, 95% CI 1.01-4.38, p = 0.048), UC diagnosis at an older age (OR 2.23, 95% CI 1.03-4.83, p = 0.043) and appendectomy prior to UC diagnosis (OR 2.66, 95% CI 1.06-6.71, p = 0.038) were independent risk factors for AN. Use of thiopurines (OR 0.21, 95% CI 0.06-0.74, p = 0.015) and being in a surveillance colonoscopy programme (OR 0.33; 95% CI 0.16-0.67; p = 0.002) were independent protective factors for AN. The risk of AN among UC patients is lower than previously reported but steadily increases from the time of UC diagnosis. The widespread use of thiopurines may have influenced this reduced incidence of UC-related neoplasias. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

PubMed

Al-Hadid, Qais; White, Jonelle; Clarke, Steven

2016-02-12

A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.

PubMed

Panikar, Chitra S; Rajpathak, Shriram N; Abhyankar, Varada; Deshmukh, Saniya; Deobagkar, Deepti D

2015-12-01

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

Beta-adrenergic control of phosphatidylcholine synthesis by transmethylation in hepatocytes from juvenile, adult and adrenalectomized rats.

PubMed Central

Marin-Cao, D; Alvarez Chiva, V; Mato, J M

1983-01-01

Changes in isoprenaline-sensitive phospholipid methyltransferase were studied in hepatocytes isolated from juvenile, mature and adrenalectomized rats. Isoprenaline produced greater stimulation of cyclic AMP accumulation in juvenile and mature adrenalectomized rats than in mature animals. Similarly, isoprenaline stimulated phospholipid methyltransferase in juvenile and mature adrenalectomized rats but had no effect in mature animals. Isoprenaline-mediated activation of phospholipid methyltransferase in adrenalectomized rats was time- and dose-dependent. In hepatocytes isolated from adrenalectomized rats incubated with [Me-3H]methionine or [3H]-ethanolamine the addition of isoprenaline increased the amount of radioactivity incorporated into phosphatidylcholine. The activation by isoprenaline of phospholipid methyltransferase was abolished by the beta-blocker propranolol and by insulin. These results indicate that rat liver the occupation of functional beta-receptors causes a stimulation of phospholipid methylation. It is suggested that, as reported previously, cyclic AMP activates phospholipid methyltransferase. PMID:6320796

Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

PubMed

Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

2010-09-20

KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation.

PubMed

Thanh Mai Pham, Le; Kim, Yong Hwan

2016-01-01

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5'-adenosyl)-L-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones. Copyright © 2015 Elsevier Inc. All rights reserved.

Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimura, Shuhei; Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574; Sawatsubashi, Shun

2008-07-11

Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressedmore » EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.« less

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

PubMed Central

Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2015-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia.

PubMed

Tanaka, Yoichi; Kato, Motohiro; Hasegawa, Daisuke; Urayama, Kevin Y; Nakadate, Hisaya; Kondoh, Kensuke; Nakamura, Kozue; Koh, Katsuyoshi; Komiyama, Takako; Manabe, Atsushi

2015-10-01

Genotyping of TPMT prior to 6-mercaptopurine (6-MP) administration in acute lymphoblastic leukaemia (ALL) patients has been integrated into clinical practice in some populations of European ancestry. However, the comparable rates of 6-MP myelotoxicity, but rarity of TPMT variants, in Asians suggest that major determinants have yet to be discovered in this population. We genotyped 92 Japanese paediatric ALL patients for NUDT15 rs116855232, a 6-MP toxicity-related locus discovered in Asians. Logistic regression and survival analysis were used to evaluate its association with leucopenia, hepatotoxicity, 6-MP dose reduction, therapy interruption and event-free survival. The allele frequency of rs116855232 was 0·16, and leucopenia was more common in carriers of the T allele (odds ratio, 7·20; 95% confidence interval, 2·49-20·80; P = 2·7 × 10(-4) ). As leucopenia results in 6-MP dose reduction, we observed average doses during maintenance therapy of 40·7, 29·3 and 8·8 mg/m(2) for patients with CC, CT and TT genotypes, respectively (P < 0·001). Hepatotoxicity was observed only in CC genotype patients. Event-free survival did not significantly differ by NUDT15 genotype. rs116855232 is an important determinant of 6-MP myelotoxicity in Japanese children with ALL and may represent the most robust toxicity-related locus in Asians to date. Considerations for clinical application may be warranted. © 2015 John Wiley & Sons Ltd.

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

PubMed

McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2016-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity

PubMed Central

Jacques-Fricke, Bridget T.; Gammill, Laura S.

2014-01-01

Neural crest precursors express genes that cause them to become migratory, multipotent cells, distinguishing them from adjacent stationary neural progenitors in the neurepithelium. Histone methylation spatiotemporally regulates neural crest gene expression; however, the protein methyltransferases active in neural crest precursors are unknown. Moreover, the regulation of methylation during the dynamic process of neural crest migration is unclear. Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells. NSD3 expression commences before up-regulation of neural crest genes, and NSD3 is necessary for expression of the neural plate border gene Msx1, as well as the key neural crest transcription factors Sox10, Snail2, Sox9, and FoxD3, but not gene expression generally. Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation. In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration. These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration. PMID:25318671

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

PubMed Central

Levac, Dylan; Cázares, Paulo; Yu, Fang

2016-01-01

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

PubMed

Zhang, Xiaohong; Jeffs, Graham; Ren, Xiaolin; O'Donovan, Peter; Montaner, Beatriz; Perrett, Conal M; Karran, Peter; Xu, Yao-Zhong

2007-03-01

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

A simplified characterization of S-adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting† †Electronic supplementary information (ESI) available: Experimental materials and methods, characterization of all compounds (1H–1H COSY NMR, 1H–13C edited HSQC NMR, MS analysis), supplementary figures and tables, worksheets for the 1-Step EZ-MTase assay using both UV- and fluorescence-detection mode, a worksheet for the determination of glycine N-methyltransferase activity within biological samples. Samples of purified TM0936 will be distributed upon request. See DOI: 10.1039/c7sc02830j

PubMed Central

Walters, Ryan O.; Huffman, Derek M.

2017-01-01

Methyltransferases use S-adenosyl-l-methionine (SAM) to deposit methyl marks. Many of these epigenetic ‘writers’ are associated with gene regulation. As cancer etiology is highly correlated with misregulated methylation patterns, methyltransferases are emerging therapeutic targets. Successful assignment of methyltransferases' roles within intricate biological networks relies on (1) the access to enzyme mechanistic insights and (2) the efficient screening of chemical probes against these targets. To characterize methyltransferases in vitro and in vivo, we report a highly-sensitive one-step deaminase-linked continuous assay where the S-adenosyl-l-homocysteine (SAH) enzyme-product is rapidly and quantitatively catabolized to S-inosyl-l-homocysteine (SIH). To highlight the broad capabilities of this assay, we established enzymatic characteristics of two protein arginine methyltransferases (PRMT5 and PRMT7), a histone-lysine N-methyltransferase (DIM-5) and a sarcosine/dimethylglycine N-methyltransferase (SDMT). Since the coupling deaminase TM0936 displays robust activity over a broad pH-range we determined the pH dependence of SDMT reaction rates. TM0936 reactions are monitored at 263 nm, so a drawback may arise when methyl acceptor substrates absorb within this UV-range. To overcome this limitation, we used an isosteric fluorescent SAM-analog: S-8-aza-adenosyl-l-methionine. Most enzymes tolerated this probe and sustained methyltransfers were efficiently monitored through loss of fluorescence at 360 nm. Unlike discontinuous radioactive- and antibody-based assays, our assay provides a simple, versatile and affordable approach towards the characterization of methyltransferases. Supported by three logs of linear dynamic range, the 1-Step EZ-MTase can detect methylation rates as low as 2 μM h–1, thus making it possible to quantify low nanomolar concentrations of glycine N-methyltransferase within crude biological samples. With Z′-factors above 0.75, this assay is well suited to high-throughput screening and may promote the identification of novel therapeutics. PMID:29449933

Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

PubMed Central

Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

2016-01-01

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site.

PubMed

van Bemmel, Dana M; Brank, Adam S; Eritja, Ramon; Marquez, Victor E; Christman, Judith K

2009-09-15

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

PubMed Central

van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

2009-01-01

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

PubMed

Horowitz, Scott; Dirk, Lynnette M A; Yesselman, Joseph D; Nimtz, Jennifer S; Adhikari, Upendra; Mehl, Ryan A; Scheiner, Steve; Houtz, Robert L; Al-Hashimi, Hashim M; Trievel, Raymond C

2013-10-16

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

PubMed

Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

2017-01-01

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Inherited NUDT15 Variant Is a Genetic Determinant of Mercaptopurine Intolerance in Children With Acute Lymphoblastic Leukemia

PubMed Central

Yang, Jun J.; Landier, Wendy; Yang, Wenjian; Liu, Chengcheng; Hageman, Lindsey; Cheng, Cheng; Pei, Deqing; Chen, Yanjun; Crews, Kristine R.; Kornegay, Nancy; Wong, F. Lennie; Evans, William E.; Pui, Ching-Hon; Bhatia, Smita; Relling, Mary V.

2015-01-01

Purpose Mercaptopurine (MP) is the mainstay of curative therapy for acute lymphoblastic leukemia (ALL). We performed a genome-wide association study (GWAS) to identify comprehensively the genetic basis of MP intolerance in children with ALL. Patients and Methods The discovery GWAS and replication cohorts included 657 and 371 children from two prospective clinical trials. MP dose intensity was a marker for drug tolerance and toxicities and was defined as prescribed dose divided by the planned protocol dose during maintenance therapy; its association with genotype was evaluated using a linear mixed-effects model. Results MP dose intensity varied by race and ethnicity and was negatively correlated with East Asian genetic ancestry (P < .001). The GWAS revealed two genome-wide significant loci associated with dose intensity: rs1142345 in TPMT (Tyr240Cys, present in *3A and *3C variants; P = 8.6 × 10−9) and rs116855232 in NUDT15 (P = 8.8 × 10−9), with independent replication. Patients with TT genotype at rs116855232 were exquisitely sensitive to MP, with an average dose intensity of 8.3%, compared with those with TC and CC genotypes, who tolerated 63% and 83.5% of the planned dose, respectively. The NUDT15 variant was most common in East Asians and Hispanics, rare in Europeans, and not observed in Africans, contributing to ancestry-related differences in MP tolerance. Of children homozygous for either TPMT or NUDT15 variants or heterozygous for both, 100% required ≥ 50% MP dose reduction, compared with only 7.7% of others. Conclusion We describe a germline variant in NUDT15 strongly associated with MP intolerance in childhood ALL, which may have implications for treatment individualization in this disease. PMID:25624441

Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia.

PubMed

Yang, Jun J; Landier, Wendy; Yang, Wenjian; Liu, Chengcheng; Hageman, Lindsey; Cheng, Cheng; Pei, Deqing; Chen, Yanjun; Crews, Kristine R; Kornegay, Nancy; Wong, F Lennie; Evans, William E; Pui, Ching-Hon; Bhatia, Smita; Relling, Mary V

2015-04-10

Mercaptopurine (MP) is the mainstay of curative therapy for acute lymphoblastic leukemia (ALL). We performed a genome-wide association study (GWAS) to identify comprehensively the genetic basis of MP intolerance in children with ALL. The discovery GWAS and replication cohorts included 657 and 371 children from two prospective clinical trials. MP dose intensity was a marker for drug tolerance and toxicities and was defined as prescribed dose divided by the planned protocol dose during maintenance therapy; its association with genotype was evaluated using a linear mixed-effects model. MP dose intensity varied by race and ethnicity and was negatively correlated with East Asian genetic ancestry (P < .001). The GWAS revealed two genome-wide significant loci associated with dose intensity: rs1142345 in TPMT (Tyr240Cys, present in *3A and *3C variants; P = 8.6 × 10(-9)) and rs116855232 in NUDT15 (P = 8.8 × 10(-9)), with independent replication. Patients with TT genotype at rs116855232 were exquisitely sensitive to MP, with an average dose intensity of 8.3%, compared with those with TC and CC genotypes, who tolerated 63% and 83.5% of the planned dose, respectively. The NUDT15 variant was most common in East Asians and Hispanics, rare in Europeans, and not observed in Africans, contributing to ancestry-related differences in MP tolerance. Of children homozygous for either TPMT or NUDT15 variants or heterozygous for both, 100% required ≥ 50% MP dose reduction, compared with only 7.7% of others. We describe a germline variant in NUDT15 strongly associated with MP intolerance in childhood ALL, which may have implications for treatment individualization in this disease. © 2015 by American Society of Clinical Oncology.

Monomethylioarsenicals are substratres for human arsenic (+3 oxidation state) methyltransferase

EPA Science Inventory

Monomethylthioarsenicals are substrates for human arsenic (+3 oxida1tion state) methyltransferase Methylated thioarsenicals are structural analogs of methylated oxyarsenic in which one or more oxygen atom bound t...

Monolignol 4-O-methyltransferases and uses thereof

DOEpatents

Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

2014-11-18

Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

Properly Substituted Analogues of BIX-01294 Lose Inhibition of G9a Histone Methyltransferase and Gain Selective Anti-DNA Methyltransferase 3A Activity

PubMed Central

Rotili, Dante; Tarantino, Domenico; Marrocco, Biagina; Gros, Christina; Masson, Véronique; Poughon, Valérie; Ausseil, Fréderic; Chang, Yanqi; Labella, Donatella; Cosconati, Sandro; Di Maro, Salvatore; Novellino, Ettore; Schnekenburger, Michael; Grandjenette, Cindy; Bouvy, Celine; Diederich, Marc; Cheng, Xiaodong; Arimondo, Paola B.; Mai, Antonello

2014-01-01

Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency. PMID:24810902

Identification of phosphomethylethanolamine N-methyltransferase from Arabidopsis and its role in choline and phospholipid metabolism.

PubMed

BeGora, Michael D; Macleod, Mitchell J R; McCarry, Brian E; Summers, Peter S; Weretilnyk, Elizabeth A

2010-09-17

Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-l-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.

Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

PubMed Central

Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

2016-01-01

Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

PubMed Central

Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

2010-01-01

N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

PubMed

Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

2007-02-23

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

PubMed

Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

2017-06-01

Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Metadynamics Simulation Study on the Conformational Transformation of HhaI Methyltransferase: An Induced-Fit Base-Flipping Hypothesis

PubMed Central

Ye, Fei; Zhao, Dan; Chen, Shijie; Jiang, Ren-Wang; Jiang, Hualiang; Luo, Cheng

2014-01-01

DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an “induced-fit” process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators. PMID:25045662

Photo-oxidation of 6-thioguanine by UVA: the formation of addition products with low molecular weight thiol compounds.

PubMed

Ren, Xiaolin; Xu, Yao-Zhong; Karran, Peter

2010-01-01

The thiopurine, 6-thioguanine (6-TG) is present in the DNA of patients treated with the immunosuppressant and anticancer drugs azathioprine or mercaptopurine. The skin of these patients is selectively sensitive to UVA radiation-which comprises >90% of the UV light in incident sunlight-and they suffer high rates of skin cancer. UVA irradiation of DNA 6-TG produces DNA lesions that may contribute to the development of cancer. Antioxidants can protect 6-TG against UVA but 6-TG oxidation products may undergo further reactions. We characterize some of these reactions and show that addition products are formed between UVA-irradiated 6-TG and N-acetylcysteine and other low molecular weight thiol compounds including β-mercaptoethanol, cysteine and the cysteine-containing tripeptide glutathione (GSH). GSH is also adducted to 6-TG-containing oligodeoxynucleotides in an oxygen- and UVA-dependent nucleophilic displacement reaction that involves an intermediate oxidized 6-TG, guanine sulfonate (G(SO3) ). These photochemical reactions of 6-TG, particularly the formation of a covalent oligodeoxynucleotide-GSH complex, suggest that crosslinking of proteins or low molecular weight thiol compounds to DNA may be a previously unrecognized hazard in sunlight-exposed cells of thiopurine-treated patients. © 2010 The Authors. Journal Compilation. The American Society of Photobiology.

Chemical Probes of Histone Lysine Methyltransferases

PubMed Central

2015-01-01

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin.

PubMed

Witek, Marta A; Conn, Graeme L

2014-09-01

The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m(1)A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m(1)A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m(1)A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical enzyme is a functional 16S rRNA (m(1)A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

PubMed

Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

2017-04-01

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polymorphisms in arsenic(+III oxidation state) methyltransferase (AS3MT) predict gene expression of AS3MT as well as arsenic metabolism.

PubMed

Engström, Karin; Vahter, Marie; Mlakar, Simona Jurkovic; Concha, Gabriela; Nermell, Barbro; Raqib, Rubhana; Cardozo, Alejandro; Broberg, Karin

2011-02-01

Arsenic (As) occurs as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in humans, and the methylation pattern demonstrates large interindividual differences. The fraction of urinary MMA is a marker for susceptibility to As-related diseases. We evaluated the impact of polymorphisms in five methyltransferase genes on As metabolism in two populations, one in South America and one in Southeast Asia. The methyltransferase genes were arsenic(+III oxidation state) methyltransferase (AS3MT), DNA-methyltransferase 1a and 3b (DNMT1a and DNMT3b, respectively), phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine methyltransferase (BHMT). AS3MT expression was analyzed in peripheral blood. Subjects were women exposed to As in drinking water in the Argentinean Andes [n = 172; median total urinary As (U-As), 200 µg/L] and in rural Bangladesh (n = 361; U-As, 100 µg/L; all in early pregnancy). Urinary As metabolites were measured by high-pressure liquid chromatography/inductively coupled plasma mass spectrometry. Polymorphisms (n = 22) were genotyped with Sequenom, and AS3MT expression was measured by quantitative real-time polymerase chain reaction using TaqMan expression assays. Six AS3MT polymorphisms were significantly associated with As metabolite patterns in both populations (p ≤ 0.01). The most frequent AS3MT haplotype in Bangladesh was associated with a higher percentage of MMA (%MMA), and the most frequent haplotype in Argentina was associated with a lower %MMA and a higher percentage of DMA. Four polymorphisms in the DNMT genes were associated with metabolite patterns in Bangladesh. Noncoding AS3MT polymorphisms affected gene expression of AS3MT in peripheral blood, demonstrating that one functional impact of AS3MT polymorphisms may be altered levels of gene expression. Polymorphisms in AS3MT significantly predicted As metabolism across these two very different populations, suggesting that AS3MT may have an impact on As metabolite patterns in populations worldwide.

Maintenance therapy of childhood acute lymphoblastic leukemia revisited-Should drug doses be adjusted by white blood cell, neutrophil, or lymphocyte counts?

PubMed

Schmiegelow, Kjeld; Nersting, Jacob; Nielsen, Stine Nygaard; Heyman, Mats; Wesenberg, Finn; Kristinsson, Jon; Vettenranta, Kim; Schrøeder, Henrik; Weinshilboum, Richard; Jensen, Katrine Lykke; Grell, Kathrine; Rosthoej, Susanne

2016-12-01

6-Mercaptopurine (6MP) and methotrexate (MTX) based maintenance therapy is a critical phase of childhood acute lymphoblastic leukemia treatment. Wide interindividual variations in drug disposition warrant frequent doses adjustments, but there is a lack of international consensus on dose adjustment guidelines. To identify relapse predictors, we collected 28,255 data sets on drug doses and blood counts (median: 47/patient) and analyzed erythrocyte (Ery) levels of cytotoxic 6MP/MTX metabolites in 9,182 blood samples (median: 14 samples/patient) from 532 children on MTX/6MP maintenance therapy targeted to a white blood cell count (WBC) of 1.5-3.5 × 10 9 /l. After a median follow-up of 13.8 years for patients in remission, stepwise Cox regression analysis did not find age, average doses of 6MP and MTX, hemoglobin, absolute lymphocyte counts, thrombocyte counts, or Ery levels of 6-thioguanine nucleotides or MTX (including its polyglutamates) to be significant relapse predictors. The parameters significantly associated with risk of relapse (N = 83) were male sex (hazard ratio [HR] 2.0 [1.3-3.1], P = 0.003), WBC at diagnosis (HR = 1.04 per 10 × 10 9 /l rise [1.00-1.09], P = 0.048), the absolute neutrophil count (ANC; HR = 1.7 per 10 9 /l rise [1.3-2.4], P = 0.0007), and Ery thiopurine methyltransferase activity (HR = 2.7 per IU/ml rise [1.1-6.7], P = 0.03). WBC was significantly related to ANC (Spearman correlation coefficient, r s  = 0.77; P < 0.001), and only a borderline significant risk factor for relapse (HR = 1.28 [95% CI: 1.00-1.64], P = 0.046) when ANC was excluded from the Cox model. This study indicates that a low neutrophil count is likely to be the best hematological target for dose adjustments of maintenance therapy. © 2016 Wiley Periodicals, Inc.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yuan; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3; Wu, Keqiang

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants.more » We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.« less

Structural characterization of the mitomycin 7-O-methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Shanteri; Chang, Aram; Goff, Randal D.

2014-10-02

Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylationmore » to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.« less

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.

PubMed

Gonsalvez, Graydon B; Tian, Liping; Ospina, Jason K; Boisvert, François-Michel; Lamond, Angus I; Matera, A Gregory

2007-08-27

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

PubMed Central

Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

2007-01-01

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian P

2015-01-02

Ergothioneine is an N-α-trimethyl-2-thiohistidine derivative that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox-active betaine starts with trimethylation of the α-amino group of histidine. The three consecutive methyl transfers are catalyzed by the S-adenosylmethionine-dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N-α-dimethylhistidine and S-adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 10(7)-fold and transform the histidine-methyltransferase into a proficient tryptophan-methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

PubMed

Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

2018-01-01

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

PubMed Central

Nield, Blair S.; Willows, Robert D.; Torda, Andrew E.; Gillings, Michael R.; Holmes, Andrew J.; Nevalainen, K.M. Helena; Stokes, H.W.; Mabbutt, Bridget C.

2004-01-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%–28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins. PMID:15152095

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase.

PubMed

Nield, Blair S; Willows, Robert D; Torda, Andrew E; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Mabbutt, Bridget C

2004-06-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes▿

PubMed Central

Schilhabel, Anke; Studenik, Sandra; Vödisch, Martin; Kreher, Sandra; Schlott, Bernhard; Pierik, Antonio Y.; Diekert, Gabriele

2009-01-01

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases. PMID:19011025

Modification of lignin content and composition in plants

DOEpatents

Ye, Zheng-Hua

2002-01-01

Plants and methods of preparing plants having reduced lignin content and/or altered lignin composition are provided. The activities of caffeoyl-CoA O-methyltransferase and/or caffeic acid O-methyltransferase enzymes in the modified plants are reduced.

Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina

2017-08-15

Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

Yeast proteins Gar1p, Nop1p, Npl3p, Nsr1p, and Rps2p are natively methylated and are substrates of the arginine methyltransferase Hmt1p.

PubMed

Yagoub, Daniel; Hart-Smith, Gene; Moecking, Jonas; Erce, Melissa A; Wilkins, Marc R

2015-09-01

The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Site-specific bioalkylation of rapamycin by the RapM 16-O-methyltransferase.

PubMed

Law, Brian J C; Struck, Anna-Winona; Bennett, Matthew R; Wilkinson, Barrie; Micklefield, Jason

2015-05-01

The methylation of natural products by S -adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors in vitro and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple S -ethyl or S -allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches.

Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis

EPA Science Inventory

Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt), yielding mono- , di- , and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation,...

Structure and function of flavivirus NS5 methyltransferase.

PubMed

Zhou, Yangsheng; Ray, Debashish; Zhao, Yiwei; Dong, Hongping; Ren, Suping; Li, Zhong; Guo, Yi; Bernard, Kristen A; Shi, Pei-Yong; Li, Hongmin

2007-04-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

A Sensitive Luminescent Assay for the Histone Methyltransferase NSD1 and Other SAM-Dependent Enzymes

PubMed Central

Drake, Katherine M.; Watson, Venita G.; Kisielewski, Anne; Glynn, Rebecca

2014-01-01

Abstract A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias. PMID:24927133

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

DOE PAGES

Ticak, Tomislav; Kountz, D. J.; Girosky, K. E.; ...

2014-10-13

COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptorsmore » such as nitrate or fumarate, producing dimethylglycine and CO 2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. Additionally, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. Lastly, they are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.« less

The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

PubMed

Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

2014-08-01

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. Copyright © 2014 by the Genetics Society of America.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases.

PubMed

Yang, Ya-Ling; Wang, Feng-Sheng; Li, Sung-Chou; Tiao, Mao-Meng; Huang, Ying-Hsien

2017-01-18

MicroRNA-29 (miR-29) is found to modulate hepatic stellate cells' (HSCs) activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to bile duct-ligation (BDL) to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A) expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis

EPA Science Inventory

The biotransformation of inorganic arsenic (iAs) involves methylation by an arsenic (+3 oxidation state) methyltransferase (AS3MT), yielding methyl arsenic (MA), dimethyl arsenic (DMA), and trimethylarsenic (TMA). To identify molecular mechanisms that coordinate arsenic biotra...

Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culman, J.; Torda, T.; Weise, V.K.

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments ofmore » other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.« less

Discovery of reversible DNA methyltransferase and lysine methyltransferase G9a inhibitors with antitumoral in vivo efficacy.

PubMed

Rabal, Obdulia; San José-Eneriz, Edurne; Agirre, Xabier; Sánchez-Arias, Juan A; Vilas-Zornoza, Amaia; Ugarte, Ana; De Miguel, Irene; Miranda, Estibaliz; Garate, Leire; Fraga, Mario; Santamarina, Pablo; Fernandez Perez, Raul; Ordoñez, Raquel; Sáez, Elena; Roa, Sergio; Garcia-Barchino, Maria Jose; Martinez-Climent, Jose Angel; Liu, Yingying; Wu, Wei; Xu, Musheng; Prosper, Felipe; Oyarzabal, Julen

2018-06-28

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear anti-proliferative efficacies (GI50 values in the nanomolar range). Based on epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept (ref 53). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human AML (Acute Myeloid Leukemia) xenograft in a mouse model.

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.

2010-07-23

Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerstermore » resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.« less

Mechanism of activation of methyltransferases involved in translation by the Trm112 'hub' protein.

PubMed

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-08-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

PubMed Central

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H.; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-01-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases. PMID:21478168

Evolution of novel O-methyltransferases from the Vanilla planifolia caffeic acid O-methyltransferase.

PubMed

Li, Huaijun Michael; Rotter, David; Hartman, Thomas G; Pak, Fulya E; Havkin-Frenkel, Daphna; Belanger, Faith C

2006-06-01

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.

Polymorphisms in Arsenic(+III Oxidation State) Methyltransferase (AS3MT) Predict Gene Expression of AS3MT as Well as Arsenic Metabolism

PubMed Central

Engström, Karin; Vahter, Marie; Mlakar, Simona Jurkovic; Concha, Gabriela; Nermell, Barbro; Raqib, Rubhana; Cardozo, Alejandro; Broberg, Karin

2011-01-01

Background Arsenic (As) occurs as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in humans, and the methylation pattern demonstrates large interindividual differences. The fraction of urinary MMA is a marker for susceptibility to As-related diseases. Objectives We evaluated the impact of polymorphisms in five methyltransferase genes on As metabolism in two populations, one in South America and one in Southeast Asia. The methyltransferase genes were arsenic(+III oxidation state) methyltransferase (AS3MT), DNA-methyltransferase 1a and 3b (DNMT1a and DNMT3b, respectively), phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine methyltransferase (BHMT). AS3MT expression was analyzed in peripheral blood. Methods Subjects were women exposed to As in drinking water in the Argentinean Andes [n = 172; median total urinary As (U-As), 200 μg/L] and in rural Bangladesh (n = 361; U-As, 100 μg/L; all in early pregnancy). Urinary As metabolites were measured by high-pressure liquid chromatography/inductively coupled plasma mass spectrometry. Polymorphisms (n = 22) were genotyped with Sequenom, and AS3MT expression was measured by quantitative real-time polymerase chain reaction using TaqMan expression assays. Results Six AS3MT polymorphisms were significantly associated with As metabolite patterns in both populations (p ≤ 0.01). The most frequent AS3MT haplotype in Bangladesh was associated with a higher percentage of MMA (%MMA), and the most frequent haplotype in Argentina was associated with a lower %MMA and a higher percentage of DMA. Four polymorphisms in the DNMT genes were associated with metabolite patterns in Bangladesh. Noncoding AS3MT polymorphisms affected gene expression of AS3MT in peripheral blood, demonstrating that one functional impact of AS3MT polymorphisms may be altered levels of gene expression. Conclusions Polymorphisms in AS3MT significantly predicted As metabolism across these two very different populations, suggesting that AS3MT may have an impact on As metabolite patterns in populations worldwide. PMID:21247820

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

*Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate ciona intestinalis

EPA Science Inventory

Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt) , yielding mono-, di-, and trimethylated arsenicals. A comparative genomic approach focused on Ciona intestinaJis, an invertebrate chordate, was u...

DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

EPA Science Inventory

Diversity of arsenic metabolism in cultured human cancer cell lines.

Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

PubMed

Akhberdi, Oren; Zhang, Qian; Wang, Haichuan; Li, Yingying; Chen, Longfei; Wang, Dan; Yu, Xi; Wei, Dongsheng; Zhu, Xudong

2018-05-01

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi. Copyright © 2018 Elsevier GmbH. All rights reserved.

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

PubMed

Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

2012-08-01

Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV. Copyright © 2012 Elsevier B.V. All rights reserved.

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals

PubMed Central

Thomas, David J.; Li, Jiaxin; Waters, Stephen B.; Xing, Weibing; Adair, Blakely M.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav

2008-01-01

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme’s role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway. PMID:17202581

A Continuous, Quantitative Fluorescent Assay for Plant Caffeic acid O-Methyltransferases

USDA-ARS?s Scientific Manuscript database

Plant caffeic acid O-methyltransferases (COMTs) use s-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in-vivo, and will generally utilize a range of phenolic compounds in-vitro. Collazo et al. (2005; Analytical Biochemistry 342: 86-92) have pu...

Mouse arsenic (+3 oxidation state) methyltransferase genotype affects metabolism and tissue dosimetry of arsenicals after arsenite administration in drinking water

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes methylation of inorganic arsenic producing a number of methylated arsenic metabolites. Although methylation has been commonly considered a pathway for detoxification of arsenic, some highly reactive methylated ars...

RmtA, a putative arginine methyltransferase, regulates secondary metabolism and development in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Aspergillus flavus is found colonizing numerous oil seed crops such as corn, peanuts, sorghum, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been de...

Comparative Distribution and Retention of Arsenic in Arsenic (+3 Oxidation State) Methyltransferase Knockout and Wild Type Mice

EPA Science Inventory

The mouse arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a ~ 43 kDa protein that catalyzes conversion of inorganic arsenic into methylated products. Heterologous expression of AS3MT or its silencing by RNA interference controls arsenic methylation phenotypes...

Cell Line Controls for the Genotyping of a Spectrum of Human Single Nucleotide Polymorphisms in the Clinical Laboratory.

PubMed

Kimbacher, Christine; Paar, Christian; Freystetter, Andrea; Berg, Joerg

2018-05-01

Genotyping for clinically important single nucleotide polymorphisms (SNPs) is performed by many clinical routine laboratories. To support testing, quality controls and reference materials are needed. Those may be derived from residual patient samples, left over samples of external quality assurance schemes, plasmid DNA or DNA from cell lines. DNAs from cell lines are commutable and available in large amounts. DNA from 38 cell lines were examined for suitability as controls in 11 SNP assays that are frequently used in a clinical routine laboratory: FV (1691G>A), FII (20210G>A), PAI-1 4G/5G polymorphism, MTHFR (677C>T, 1298A>C), HFE (H63D, S65C, C282Y), APOE (E2, E3, E4), LPH (-13910C>T), UGT1A1 (*28, *36, *37), TPMT (*2, *3A, *3B, *3C), VKORC1 (-1639G>A, 1173C>T), CYP2C9 (*2, *3, *5). Genotyping was performed by real-time PCR with melting curve analysis and confirmed by bi-directional sequencing. We find an almost complete spectrum of genotypic constellations within these 38 cell lines. About 12 cell lines appear sufficient as genotypic controls for the 11 SNP assays by covering almost all of the genotypes. However, hetero- and homozygous genotypes for FII and the alleles TPMT*2, UGT1A1*37 and CYP2C9*5 were not detected in any of the cell lines. DNA from most of the examined cell lines appear suitable as quality controls for these SNP assays in the laboratory routine, as to the implementation of those assays or to prepare samples for quality assurance schemes. Our study may serve as a pilot to further characterize these cell lines to arrive at the status of reference materials.

Crohn's disease management after intestinal resection: a randomised trial.

PubMed

De Cruz, Peter; Kamm, Michael A; Hamilton, Amy L; Ritchie, Kathryn J; Krejany, Efrosinia O; Gorelik, Alexandra; Liew, Danny; Prideaux, Lani; Lawrance, Ian C; Andrews, Jane M; Bampton, Peter A; Gibson, Peter R; Sparrow, Miles; Leong, Rupert W; Florin, Timothy H; Gearry, Richard B; Radford-Smith, Graham; Macrae, Finlay A; Debinski, Henry; Selby, Warwick; Kronborg, Ian; Johnston, Michael J; Woods, Rodney; Elliott, P Ross; Bell, Sally J; Brown, Steven J; Connell, William R; Desmond, Paul V

2015-04-11

Most patients with Crohn's disease need an intestinal resection, but a majority will subsequently experience disease recurrence and require further surgery. This study aimed to identify the optimal strategy to prevent postoperative disease recurrence. In this randomised trial, consecutive patients from 17 centres in Australia and New Zealand undergoing intestinal resection of all macroscopic Crohn's disease, with an endoscopically accessible anastomosis, received 3 months of metronidazole therapy. Patients at high risk of recurrence also received a thiopurine, or adalimumab if they were intolerant to thiopurines. Patients were randomly assigned to parallel groups: colonoscopy at 6 months (active care) or no colonoscopy (standard care). We used computer-generated block randomisation to allocate patients in each centre to active or standard care in a 2:1 ratio. For endoscopic recurrence (Rutgeerts score ≥i2) at 6 months, patients stepped-up to thiopurine, fortnightly adalimumab with thiopurine, or weekly adalimumab. The primary endpoint was endoscopic recurrence at 18 months. Patients and treating physicians were aware of the patient's study group and treatment, but central reading of the endoscopic findings was undertaken blind to the study group and treatment. Analysis included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00989560. Between Oct 13, 2009, and Sept 28, 2011, 174 (83% high risk across both active and standard care groups) patients were enrolled and received at least one dose of study drug. Of 122 patients in the active care group, 47 (39%) stepped-up treatment. At 18 months, endoscopic recurrence occurred in 60 (49%) patients in the active care group and 35 (67%) patients in the standard care group (p=0.03). Complete mucosal normality was maintained in 27 (22%) of 122 patients in the active care group versus four (8%) in the standard care group (p=0.03). In the active care arm, of those with 6 months recurrence who stepped up treatment, 18 (38%) of 47 patients were in remission 12 months later; conversely, of those in remission at 6 months who did not change therapy recurrence occurred in 31 (41%) of 75 patients 12 months later. Smoking (odds ratio [OR] 2.4, 95% CI 1.2-4.8, p=0.02) and the presence of two or more clinical risk factors including smoking (OR 2.8, 95% CI 1.01-7.7, p=0.05) increased the risk of endoscopic recurrence. The incidence and type of adverse and severe adverse events did not differ significantly between patients in the active care and standard care groups (100 [82%] of 122 vs 45 [87%] of 52; p=0.51) and (33 [27%] of 122 vs 18 [35%] of 52; p=0.36), respectively. Treatment according to clinical risk of recurrence, with early colonoscopy and treatment step-up for recurrence, is better than conventional drug therapy alone for prevention of postoperative Crohn's disease recurrence. Selective immune suppression, adjusted for early recurrence, rather than routine use, leads to disease control in most patients. Clinical risk factors predict recurrence, but patients at low risk also need monitoring. Early remission does not preclude the need for ongoing monitoring. AbbVie, Gutsy Group, Gandel Philanthropy, Angior Foundation, Crohn's Colitis Australia, and the National Health and Medical Research Council. Copyright © 2015 Elsevier Ltd. All rights reserved.

Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii.

PubMed

Pampa, K J; Madan Kumar, S; Hema, M K; Kumara, Karthik; Naveen, S; Kunishima, Naoki; Lokanath, N K

2017-12-01

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

COBALAMIN- AND COBAMIDE-DEPENDENT METHYLTRANSFERASES

PubMed Central

Matthews, Rowena G.; Koutmos, Markos; Datta, Supratim

2008-01-01

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple SN2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co+1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled. PMID:19059104

CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

EPA Science Inventory

CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

EPA Science Inventory

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)

Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

Lignin and Fiber digestibility in Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase Downregulated Alfalfa

USDA-ARS?s Scientific Manuscript database

Alfalfa (Medicago sativa L.) is one of the most important forages in the United States. Increasing alfalfa fiber digestibility would improve forage management and ration formulation flexibility. Currently, growers and breeders rely on near infrared spectroscopy (NIRS) to predict forage quality tra...

Guanidinoacetate Methyltransferase (GAMT) Deficiency: Late Onset of Movement Disorder and Preserved Expressive Language

ERIC Educational Resources Information Center

O'Rourke, Declan J.; Ryan, Stephanie; Salomons, Gajja; Jakobs, Cornelis; Monavari, Ahmad; King, Mary D.

2009-01-01

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine biosynthesis, characterized by early-onset learning disability and epilepsy in most affected children. Severe expressive language delay is a constant feature even in the mildest clinical phenotypes. We report the clinical, biochemical, imaging, and treatment data of two…

CHARACTERIZATION OF HUMAN URINARY BLADDER CELL LINE UROTSA TRANSDUCED WITH RAT ASLLL-METHYLTRANSFERASE

EPA Science Inventory

In humans, the biomethylation of arsenic (As) is catalyzed by an As(III)-methyltransferase (Cyt19) and yields pentavalent and trivalent methylated arsenicals. Cyt19 activity and expression levels vary among tissues. For example, Cyt19 mRNA is not detected in UROtsa cells, a h...

A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

EPA Science Inventory

A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver Cytosol
Shan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas
S-adenosyl-L-methionine (AdoMet): ar...

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

USDA-ARS?s Scientific Manuscript database

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

USDA-ARS?s Scientific Manuscript database

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

PubMed

Pendleton, Kathryn E; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V; Xie, Yang; Tu, Benjamin P; Conrad, Nicholas K

2017-05-18

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N 6 -adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

PubMed

Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

2016-08-01

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Betaine:homocysteine methyltransferase--a new assay for the liver enzyme and its absence from human skin fibroblasts and peripheral blood lymphocytes.

PubMed

Wang, J A; Dudman, N P; Lynch, J; Wilcken, D E

1991-12-31

Chronic elevation of plasma homocysteine is associated with increased atherogenesis and thrombosis, and can be lowered by betaine (N,N,N-trimethylglycine) treatment which is thought to stimulate activity of the enzyme betaine:homocysteine methyltransferase. We have developed a new assay for this enzyme, in which the products of the enzyme-catalysed reaction between betaine and homocysteine are oxidised by performic acid before being separated and quantified by amino acid analysis. This assay confirmed that human liver contains abundant betaine:homocysteine methyltransferase (33.4 nmol/h/mg protein at 37 degrees C, pH 7.4). Chicken and lamb livers also contain the enzyme, with respective activities of 50.4 and 6.2 nmol/h/mg protein. However, phytohaemagglutinin-stimulated human peripheral blood lymphocytes and cultured human skin fibroblasts contained no detectable betaine:homocysteine methyltransferase (less than 1.4 nmol/h/mg protein), even after cells were pre-cultured in media designed to stimulate production of the enzyme. The results emphasize the importance of the liver in mediating the lowering of elevated circulating homocysteine by betaine.

Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

PubMed Central

Züst, Roland; Li, Shi-Hua; Xie, Xuping; Velumani, Sumathy; Chng, Melissa; Toh, Ying-Xiu; Zou, Jing; Dong, Hongping; Shan, Chao; Pang, Jassia; Qin, Cheng-Feng; Newell, Evan W.; Shi, Pei-Yong

2018-01-01

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. PMID:29298302

Quercetin-Induced Lifespan Extension in Podospora anserina Requires Methylation of the Flavonoid by the O-Methyltransferase PaMTH1.

PubMed

Warnsmann, Verena; Hainbuch, Saskia; Osiewacz, Heinz D

2018-01-01

Quercetin is a flavonoid that is ubiquitously found in vegetables and fruits. Like other flavonoids, it is active in balancing cellular reactive oxygen species (ROS) levels and has a cyto-protective function. Previously, a link between ROS balancing, aging, and the activity of O -methyltransferases was reported in different organisms including the aging model Podospora anserina. Here we describe a role of the S -adenosylmethionine-dependent O -methyltransferase PaMTH1 in quercetin-induced lifespan extension. We found that effects of quercetin treatment depend on the methylation state of the flavonoid. Specifically, we observed that quercetin treatment increases the lifespan of the wild type but not of the PaMth1 deletion mutant. The lifespan increasing effect is not associated with effects of quercetin on mitochondrial respiration or ROS levels but linked to the induction of the PaMth1 gene. Overall, our data demonstrate a novel role of O -methyltransferase in quercetin-induced longevity and identify the underlying pathway as part of a network of longevity assurance pathways with the perspective to intervene into mechanisms of biological aging.

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Analysis of Natural and Induced Variation in Tomato Glandular Trichome Flavonoids Identifies a Gene Not Present in the Reference Genome[W][OPEN

PubMed Central

Kim, Jeongwoon; Matsuba, Yuki; Ning, Jing; Schilmiller, Anthony L.; Hammar, Dagan; Jones, A. Daniel; Pichersky, Eran; Last, Robert L.

2014-01-01

Flavonoids are ubiquitous plant aromatic specialized metabolites found in a variety of cell types and organs. Methylated flavonoids are detected in secreting glandular trichomes of various Solanum species, including the cultivated tomato (Solanum lycopersicum). Inspection of the sequenced S. lycopersicum Heinz 1706 reference genome revealed a close homolog of Solanum habrochaites MOMT1 3′/5′ myricetin O-methyltransferase gene, but this gene (Solyc06g083450) is missing the first exon, raising the question of whether cultivated tomato has a distinct 3′ or 3′/5′ O-methyltransferase. A combination of mining genome and cDNA sequences from wild tomato species and S. lycopersicum cultivar M82 led to the identification of Sl-MOMT4 as a 3′ O-methyltransferase. In parallel, three independent ethyl methanesulfonate mutants in the S. lycopersicum cultivar M82 background were identified as having reduced amounts of di- and trimethylated myricetins and increased monomethylated myricetin. Consistent with the hypothesis that Sl-MOMT4 is a 3′ O-methyltransferase gene, all three myricetin methylation defective mutants were found to have defects in MOMT4 sequence, transcript accumulation, or 3′-O-methyltransferase enzyme activity. Surprisingly, no MOMT4 sequence is found in the Heinz 1706 reference genome sequence, and this cultivar accumulates 3-methyl myricetin and is deficient in 3′-methyl myricetins, demonstrating variation in this gene among cultivated tomato varieties. PMID:25128240

Floral benzenoid carboxyl methyltransferases: From in vitro to in planta function

PubMed Central

Effmert, Uta; Saschenbrecker, Sandra; Ross, Jeannine; Negre, Florence; Fraser, Chris M.; Noel, Joseph P.; Dudareva, Natalia; Piechulla, Birgit

2010-01-01

Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT’s three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in planta depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses might represent the ancestor for the presently existing floral genes which during evolution gained different expression profiles and encoded enzymes with the ability to accept structurally similar substrates. PMID:15946712

Structure and Function of Flavivirus NS5 Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou,Y.; Ray, D.; Zhao, Y.

2007-01-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showedmore » a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.« less

Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

PubMed Central

2012-01-01

Background Silene latifolia and its pollinator, the noctuid moth Hadena bicruris, represent an open nursery pollination system wherein floral volatiles, especially veratrole (1, 2-dimethoxybenzene), lilac aldehydes, and phenylacetaldehyde are of key importance for floral signaling. Despite the important role of floral scent in ensuring reproductive success in S. latifolia, the molecular basis of scent biosynthesis in this species has not yet been investigated. Results We isolated two full-length cDNAs from S. latifolia that show similarity to rose orcinol O-methyltransferase. Biochemical analysis showed that both S. latifolia guaiacol O-methyltransferase1 (SlGOMT1) &S. latifolia guaiacol O-methyltransferase2 (SlGOMT2) encode proteins that catalyze the methylation of guaiacol to form veratrole. A large Km value difference between SlGOMT1 (~10 μM) and SlGOMT2 (~501 μM) resulted that SlGOMT1 is 31-fold more catalytically efficient than SlGOMT2. qRT-PCR expression analysis showed that the SlGOMT genes are specifically expressed in flowers and male S. latifolia flowers had 3- to 4-folds higher level of GOMT gene transcripts than female flower tissues. Two related cDNAs, S. dioica O-methyltransferase1 (SdOMT1) and S. dioica O-methyltransferase2 (SdOMT2), were also obtained from the sister species Silene dioica, but the proteins they encode did not methylate guaiacol, consistent with the lack of veratrole emission in the flowers of this species. Our evolutionary analysis uncovered that SlGOMT1 and SlGOMT2 genes evolved under positive selection, whereas SdOMT1 and SdOMT2 genes show no evidence for selection. Conclusions Altogether, we report the identification and functional characterization of the gene, SlGOMT1 that efficiently catalyzes veratrole formation, whereas another copy of this gene with only one amino acid difference, SlGOMT2 was found to be less efficient for veratrole synthesis in S. latifolia. PMID:22937972

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

PubMed Central

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-01-01

Abstract Background Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required. PMID:29635374

Determination of the order of substrate addition to MspI DNA methyltransferase using a novel mechanism-based inhibitor.

PubMed Central

Taylor, C; Ford, K; Connolly, B A; Hornby, D P

1993-01-01

The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase [Som and Friedman (1991) J. Biol. Chem. 266, 2937-2945], methylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases. Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described. These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide. However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded. Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction. Images Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:8484730

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

PubMed

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-03-01

Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.

[Pharmacogenomics in routine medical care].

PubMed

Rosskopf, D; Meyer zu Schwabedissen, H E; Kroemer, H K; Siegmund, W

2010-01-01

Pharmacogenomics investigates inherited differences in drug responses including beneficial and adverse reactions. While a considerable amount of evidence for genetic influences on drug responses has been accumulated within the last decade, predominantly in small studies, its value in routine therapy is still a matter of debate. The aim of this review is to discuss well established examples where pharmacogenomic techniques can improve routine treatment. Examples include genotyping of CYP2D6 in the context of antidepressant therapy, analysis of TPMT variants for the prediction of mercaptopurine-induced bone marrow depression, VKORC1 and CYP2C9 analyses for a better control of anticoagulant administration and the SLCO1B1 variant in the context of statin-induced myopathies. Georg Thieme Verlag KG Stuttgart.New York.

Severe systemic toxicity and urinary bladder cytotoxicity and regenerative hyperplasia induced by arsenite in arsenic (+3 oxidation state) methyltransferase knockout mice. A preliminary report

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions which convert inorganic arsenic to methylated metabolites. This study determined whether the As3mt null genotype in the mouse modifies cytotoxic and proliferative effects seen in urinary bladders of wild t...

Arsenic (+3 oxidation state) methyltransferase genotype affects steady-state distribution and clearance of arsenic in arsenate-treated mice

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes formation of mono-, di-, and tri-methylated metabolites of inorganic arsenic. Distribution and retention of arsenic were compared in adult female As3mt knockout mice and wild-type C57BL/6 mice using a regimen in whi...

Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

ERIC Educational Resources Information Center

Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

2009-01-01

Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

Effect of Sodium Arsenite Dose Administered in the Drinking Water on the Urinary Bladder Epithelium of Female Arsenic (+3 oxidation state) Methyltransferase Knockout Mice

EPA Science Inventory

The enzyme arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions converting inorganic arsenic to methylated metabolites, some of which are highly cytotoxic. In a previous study, we evaluated whether the As3mt null genotype in mice modified cytotoxic and proli...

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

USDA-ARS?s Scientific Manuscript database

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

PubMed Central

Wu, Qian; Caine, Joanne M.; Thomson, Stuart A.; Slavica, Meri; Grunewald, Gary L.

2009-01-01

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT. PMID:19171483

Involvement of DNA methylation in memory processing in the honey bee.

PubMed

Lockett, Gabrielle A; Helliwell, Paul; Maleszka, Ryszard

2010-08-23

DNA methylation, an important and evolutionarily conserved epigenetic mechanism, is implicated in learning and memory processes in vertebrates, but its role in behaviour in invertebrates is unknown. We examined the role of DNA methylation in memory in the honey bee using an appetitive Pavlovian olfactory discrimination task, and by assessing the expression of DNA methyltransferase3, a key driver of epigenetic reprogramming. Here we report that DNA methyltransferase inhibition reduces acquisition retention and alters the extinction depending on treatment time, and DNA methyltransferase3 is upregulated after training. Our findings add to the understanding of epigenetic mechanisms in learning and memory, extending known roles of DNA methylation to appetitive and extinction memory, and for the first time implicate DNA methylation in memory in invertebrates.

[Definition of the specificity of DNA-methyltransferase M.Bsc4I in cell lysate by blocking of restriction endonucleases and computer modeling].

PubMed

Dedkov, V S

2009-01-01

The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.

Nonsynonymous Polymorphism in Guanine Monophosphate Synthetase Is a Risk Factor for Unfavorable Thiopurine Metabolite Ratios in Patients With Inflammatory Bowel Disease.

PubMed

Roberts, Rebecca L; Wallace, Mary C; Seinen, Margien L; van Bodegraven, Adriaan A; Krishnaprasad, Krupa; Jones, Gregory T; van Rij, Andre M; Baird, Angela; Lawrance, Ian C; Prosser, Ruth; Bampton, Peter; Grafton, Rachel; Simms, Lisa A; Studd, Corrie; Bell, Sally J; Kennedy, Martin A; Halliwell, Jacob; Gearry, Richard B; Radford-Smith, Graham; Andrews, Jane M; McHugh, Patrick C; Barclay, Murray L

2018-05-16

Up to 20% of patients with inflammatory bowel disease (IBD) who are refractory to thiopurine therapy preferentially produce 6-methylmercaptopurine (6-MMP) at the expense of 6-thioguanine nucleotides (6-TGN), resulting in a high 6-MMP:6-TGN ratio (>20). The objective of this study was to evaluate whether genetic variability in guanine monophosphate synthetase (GMPS) contributes to preferential 6-MMP metabolizer phenotype. Exome sequencing was performed in a cohort of IBD patients with 6-MMP:6-TGN ratios of >100 to identify nonsynonymous single nucleotide polymorphisms (nsSNPs). In vitro assays were performed to measure GMPS activity associated with these nsSNPs. Frequency of the nsSNPs was measured in a cohort of 530 Caucasian IBD patients. Two nsSNPs in GMPS (rs747629729, rs61750370) were detected in 11 patients with very high 6-MMP:6-TGN ratios. The 2 nsSNPs were predicted to be damaging by in silico analysis. In vitro assays demonstrated that both nsSNPs resulted in a significant reduction in GMPS activity (P < 0.05). The SNP rs61750370 was significantly associated with 6-MMP:6-TGN ratios ≥100 (odds ratio, 5.64; 95% confidence interval, 1.01-25.12; P < 0.031) in a subset of 264 Caucasian IBD patients. The GMPS SNP rs61750370 may be a reliable risk factor for extreme 6MMP preferential metabolism.

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry.

PubMed

Volk, K J; Yost, R A; Brajter-Toth, A

1989-07-14

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.

Practice Variations in the Management of Inflammatory Bowel Disease Between Pediatric and Adult Gastroenterologists.

PubMed

Assa, Amit; Avni, Irit; Ben-Bassat, Ofer; Niv, Yaron; Shamir, Raanan

2016-03-01

Different practice guidelines and consensus statements for both pediatric- and adult-onset inflammatory bowel disease exist. Although variations in medical care among gastroenterologists are common, it is unknown whether there are fundamental differences between disciplines. The primary aim of the present study was to investigate differences in common practices across disciplines. This was a prospective, questionnaire-based survey of gastroenterologists attending gastroenterology meetings in Israel. The questionnaire covered attitudes to medical resources, diagnostic and follow-up measures as well as therapeutic approaches. Overall, 120 (60%) of the approached adult gastroenterologists (AGs) and 49 (75%) pediatric gastroenterologists (PGs) completed the questionnaire. The 2 groups differed significantly in various practice areas. Pediatric patients are seen more frequently and for longer appointments. PGs tend to assess thiopurine metabolites (96% vs 47%) more often during treatment. There is a significant variation in practice between groups concerning infectious serology status and vaccinations. Methotrexate in Crohn patients is used more commonly by PGs (55% vs 22%). Long-term combination therapy of thiopurines and anti-tumor necrosis factorα agents is used more often by AGs. In patients with ulcerative colitis AGs use oral 5-aminosalicylic acid once daily (51% vs 21%) and add rectal agents from the first day of treatment (72% vs 35%) more often as compared with PGs. This study demonstrates that common practices in inflammatory bowel disease patients differ significantly between adult and pediatric practitioners. These findings call for investigating the reasons for these differences and promoting strategies to diminish these gaps.

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

PubMed Central

Mininno, Morgane; Brugière, Sabine; Gilgen, Annabelle; Ma, Sheng; Mazzoleni, Meryl; Gigarel, Océane; Martin-Laffon, Jacqueline; Ferro, Myriam; Ravanel, Stéphane

2014-01-01

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology. PMID:24748391

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation.

PubMed

Jelinic, Petar; Stehle, Jean-Christophe; Shaw, Phillip

2006-10-01

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation-DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

PubMed Central

Jelinic, Petar; Stehle, Jean-Christophe; Shaw, Phillip

2006-01-01

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation. PMID:17048991

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase.

PubMed

Debler, Erik W; Jain, Kanishk; Warmack, Rebeccah A; Feng, You; Clarke, Steven G; Blobel, Günter; Stavropoulos, Pete

2016-02-23

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

Catechol-O-Methyltransferase "Val[superscript 158]Met" Genotype, Parenting Practices and Adolescent Alcohol Use: Testing the Differential Susceptibility Hypothesis

ERIC Educational Resources Information Center

Laucht, Manfred; Blomeyer, Dorothea; Buchmann, Arlette F.; Treutlein, Jens; Schmidt, Martin H.; Esser, Gunter; Jennen-Steinmetz, Christine; Rietschel, Marcella; Zimmermann, Ulrich S.; Banaschewski, Tobias

2012-01-01

Background: Recently, first evidence has been reported for a gene-parenting interaction (G x E) with regard to adolescent alcohol use. The present investigation set out to extend this research using the catechol-O-methyltransferase ("COMT") "Val[superscript 158]Met" polymorphism as a genetic susceptibility factor. Moreover, the current study…

Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations

USDA-ARS?s Scientific Manuscript database

With S-adenosylmethionine (SAM) acting as the methyl donor, caffeic acid O-methyltransferase from Sorghum bicolor (SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde, to form sinapaldehyde. In order to determine the mechanism of SbCOMT and understand the red...

METABOLISM AND TOXICITY OF AS IN HUMAN UROTHELIAL CELLS EXPRESSING RAT ARSENIC (+3 OXIDATION STATE)-METHYLTRANSFERASE

EPA Science Inventory

The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa ce...

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

PubMed Central

Sayegh, Joyce; Clarke, Steven G.

2008-01-01

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase. PMID:18515076

Role of transmethylation reactions in alcoholic liver disease

PubMed Central

Kharbanda, Kusum K

2007-01-01

Alcoholic liver disease is a major health care problem worldwide. Findings from many laboratories, including ours, have demonstrated that ethanol feeding impairs several of the many steps involved in methionine metabolism. Ethanol consumption predominantly results in a decrease in the hepatocyte level of S-adenosylmethionine and the increases in two toxic metabolites, homocysteine and S-adenosylhomocysteine. These changes, in turn, result in serious functional consequences which include decreases in essential methylation reactions via inhibition of various methyltransferases. Of particular interest to our laboratory is the inhibition of three important enzymes, phosphatidylethanolamine methyltransferase, isoprenylcysteine carboxyl methyltransferase and protein L-isoaspartate methyltransferase. Decreased activity of these enzymes results in increased fat deposition, increased apoptosis and increased accumulation of damaged proteins-all of which are hallmark features of alcoholic liver injury. Of all the therapeutic modalities available, betaine has been shown to be the safest, least expensive and most effective in attenuating ethanol-induced liver injury. Betaine, by virtue of aiding in the remethylation of homocysteine, removes both toxic metabolites (homocysteine and S-adenosylhomocysteine), restores S-adenosylmethionine level, and reverses steatosis, apoptosis and damaged proteins accumulation. In conclusion, betaine appears to be a promising therapeutic agent in relieving the methylation and other defects associated with alcoholic abuse. PMID:17854136

O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

PubMed

Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

2017-07-01

Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario

2006-08-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed,more » purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.« less

Aurora-B Regulates RNA Methyltransferase NSUN2

PubMed Central

Sakita-Suto, Shiho; Kanda, Akifumi; Suzuki, Fumio; Sato, Sunao; Takata, Takashi

2007-01-01

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G1 phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis. PMID:17215513

Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

PubMed

James, Allison E; Rogovskyy, Artem S; Crowley, Michael A; Bankhead, Troy

2016-01-01

DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Highly Iterated Palindromic Sequences (HIPs) and Their Relationship to DNA Methyltransferases

PubMed Central

Elhai, Jeff

2015-01-01

The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences. PMID:25789551

Highly Iterated Palindromic Sequences (HIPs) and Their Relationship to DNA Methyltransferases.

PubMed

Elhai, Jeff

2015-03-17

The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences.

Purification and characterization of caffeine synthase from tea leaves.

PubMed

Kato, M; Mizuno, K; Fujimura, T; Iwama, M; Irie, M; Crozier, A; Ashihara, H

1999-06-01

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.

Noncolorectal malignancies in inflammatory bowel disease: more than meets the eye.

PubMed

Beaugerie, Laurent; Sokol, Harry; Seksik, Philippe

2009-01-01

In patients with Crohn's disease, the risk of small bowel adenocarcinoma is 20-40 times higher than the low background risk of the general population. In the subset of patients with longstanding small bowel lesions, the absolute risk of small bowel adenocarcinoma exceeds 1 per 100 patient-years after 25 years of follow-up and becomes equivalent to the risk of colorectal cancer. Growing evidence suggests that the pathogenesis of small bowel adenocarcinoma arising in inflammatory lesions of Crohn's disease is similar to that of colorectal cancer complicating chronic colonic inflammation (inflammation-dysplasia-cancer sequence). However, contrasting with the established endoscopic detection of colonic advanced neoplasias in patients with longstanding extensive colitis, there is no consensus at this time how to face the excess-risk of small bowel adenocarcinoma in patients at high risk. There are no specific clinical or imaging alert signs and endoscopic surveillance of the totality of the inflamed small bowel mucosa would suppose to perform repeated enteroscopies, with the potential limiting factor of stenosis. Very preliminary data suggest that chemoprevention with salicylates could be an alternative way for reducing the risk. Data from referral centers and from the CESAME cohort suggest that intestinal lymphomas may arise in the chronically inflamed segments in patients with inflammatory bowel disease (IBD). Regarding nonintestinal lymphomas, it is now established that IBD patients treated with thiopurines have an excess risk of lymphomas, exhibiting in most cases pathological features of lymphomas associated with immunosuppression, including the frequent presence of EBV in neoplastic tissues. There is growing evidence that treatment with thiopurines is responsible by itself for this excess risk. IBD patients receiving immunomodulators, especially young men, are also at risk (0.4 for 10,000 patient-years in the CESAME study) for developing fatal early post-mononucleosis lymphomas, like in Purtilo's syndrome, maybe in association with a background genetic susceptibility. Finally, patients receiving thiopurines and/or TNF-inhibitors are at risk for developing fatal hepatosplenic T cell lymphomas, but this risk is low (no case in the CESAME study). Whether patients receiving a monotherapy with methotrexate and/or TNF inhibitors are at increased risk of lymphomas is not known. Concordant data suggest that women receiving immunosuppressive therapy are at increased risk for developing uterine cervix dysplasia and require closer surveillance. But it is not established whether the risk of uterine cervix cancer and basal and squamous cell skin cancers (that may be associated with chronic human papillomavirus infection) is increased in patients receiving immunomodulators. Copyright 2009 S. Karger AG, Basel.

Accelerated degradation of lignin by lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium with engineered 4-O-methyltransferase from Clarkia breweri.

PubMed

Pham, Le Thanh Mai; Kim, Yong Hwan

2014-11-01

Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations. Copyright © 2014 Elsevier Inc. All rights reserved.

Methylation of Arsenic by Recombinant Human Wild-Type Arsenic (+3 Oxidation State) Methyltransferase and its Methionine 287 Threonine (M287T) Polymorph

EPA Science Inventory

ABSTRACT Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency...

Two rare cases of Epstein-Barr virus-associated lymphoproliferative disorders in inflammatory bowel disease patients on thiopurines and other immunosuppressive medications.

PubMed

Subramaniam, K; Cherian, M; Jain, S; Latimer, M; Corbett, M; D'Rozario, J; Pavli, P

2013-12-01

The setting of chronic immunosuppression in inflammatory bowel disease (IBD) may promote the proliferation of Epstein-Barr virus-positive neoplastic clones. We report two rare cases of Epstein-Barr virus-associated lymphoproliferative disorder in IBD patients: one resembled lymphomatoid granulomatosis, and the other was a lymphoma resembling Hodgkin lymphoma. There are currently no guidelines for the prevention of lymphoproliferative disorder in IBD patients on immunosuppressive therapy. © 2013 The Authors; Internal Medicine Journal © 2013 Royal Australasian College of Physicians.

Crohn's disease complicated by Epstein-Barr virus-driven haemophagocytic lymphohistiocytosis successfully treated with rituximab.

PubMed

Thompson, Grace; Pepperell, Dominic; Lawrence, Ian; McGettigan, Benjamin David

2017-02-22

We report a case of Epstein-Barr virus (EBV)-driven haemophagocytic lymphohistiocytosis (HLH) in a man with Crohn's disease treated with 6-mercaptopurine and adalimumab therapy who was successfully treated with rituximab therapy alone. This is the first published case in an adult patient with EBV-driven HLH in the setting of thiopurine use and inflammatory bowel disease to be successfully treated with rituximab therapy alone. Here, we will discuss putative immunological mechanisms which may contribute to this potentially life-threatening complication. 2017 BMJ Publishing Group Ltd.

Azathioprine-induced alopecia and leukopenia associated with NUDT15 polymorphisms.

PubMed

Nomura, H; Kurihara, Y; Saito, M; Fukushima, A; Shintani, Y; Shiiyama, R; Toshima, S; Kamata, A; Yamagami, J; Funakoshi, T; Kameyama, K; Amagai, M; Kubo, A; Umegaki-Arao, N

2018-04-28

Azathioprine (AZA), a widely used immunosuppressant, can induce cytotoxic effects including myelosuppression and alopecia. 1 Recent studies revealed that polymorphisms of NUDT15 are associated with thiopurine-induced alopecia and leukopenia. 2-5 The frequency of NUDT15 polymorphisms in East and South Asians is high (22.6% and 13.6%, respectively). 5 Thus, adverse event management during AZA treatment is essential for Asian populations with these polymorphisms. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm ofmore » the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.« less

Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum

PubMed Central

Malmquist, Nicholas A.; Moss, Thomas A.; Mecheri, Salah; Scherf, Artur; Fuchter, Matthew J.

2012-01-01

Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated Plasmodium falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC50 values of ∼100 nM, values at least 22-fold more potent than their apparent IC50 toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intraerythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-h incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a 12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration-dependent and exposure time-dependent manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit malaria parasite histone methyltransferases, resulting in rapid and irreversible parasite death. Our data position histone lysine methyltransferases as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages. PMID:23011794

A novel arsenic methyltransferase gene of Westerdykella aurantiaca isolated from arsenic contaminated soil: phylogenetic, physiological, and biochemical studies and its role in arsenic bioremediation.

PubMed

Verma, Shikha; Verma, Pankaj Kumar; Meher, Alok Kumar; Dwivedi, Sanjay; Bansiwal, Amit Kumar; Pande, Veena; Srivastava, Pankaj Kumar; Verma, Praveen Chandra; Tripathi, Rudra Deo; Chakrabarty, Debasis

2016-03-01

Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks.

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.

PubMed

Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

2011-02-04

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

PubMed Central

Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

2016-01-01

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs. PMID:26858449

Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

PubMed Central

Bayraktar, Gonca; Kreutz, Michael R.

2017-01-01

DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders. PMID:28513272

Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

DOEpatents

Liu, Chang-Jun; Cai, Yuanheng

2017-01-31

Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase | Center for Cancer Research

Cancer.gov

HHAI methyltransferase (blue ribbon) bound to oligonucleotide (strands with bonds colored yellow and green) containing a pseudorotationally constrained sugar analogue at the target position (orange bonds with cyan atoms). The south-constrained pseudosugar is rotated about its flanking phosphodiester bonds, 90° from its initial position in B-form DNA, but short of a completely

The Flexible Mind Is Associated with the Catechol-O-Methyltransferase (COMT) Val[superscript 158]Met Polymorphism: Evidence for a Role of Dopamine in the Control of Task-Switching

ERIC Educational Resources Information Center

Colzato, Lorenza S.; Waszak, Florian; Nieuwenhuis, Sander; Posthuma, Danielle; Hommel, Bernhard

2010-01-01

Genetic variability related to the catechol-O-methyltransferase (COMT) gene Val[superscript 128]Met polymorphism) has received increasing attention as a possible modulator of cognitive control functions. Recent evidence suggests that the Val[superscript 128]Met genotype may differentially affect cognitive stability and flexibility, in such a way…

Purification and Characterization of Caffeine Synthase from Tea Leaves1

PubMed Central

Kato, Misako; Mizuno, Kouichi; Fujimura, Tatsuhito; Iwama, Masanori; Irie, Masachika; Crozier, Alan; Ashihara, Hiroshi

1999-01-01

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg−1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5′-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 μm, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases. PMID:10364410

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

DOE PAGES

Hao, Yue; Blair, Patricia M.; Sharma, Abhishek; ...

2015-01-30

Peptide antibiotics represent a class of conformationally-constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here in this paper, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide amore » rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).« less

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

PubMed Central

Wang, Bo; Yu, Jianping

2015-01-01

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803. PMID:26452551

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Yue; Blair, Patricia M.; Sharma, Abhishek

Peptide antibiotics represent a class of conformationally-constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here in this paper, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide amore » rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).« less

Methylated nucleosides in tRNA and tRNA methyltransferases

PubMed Central

Hori, Hiroyuki

2014-01-01

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

Case study for the evaluation of current treatment recommendations of guanidinoacetate methyltransferase deficiency: ineffectiveness of sodium benzoate.

PubMed

Mercimek-Mahmutoglu, Saadet; Salomons, Gajja S; Chan, Alicia

2014-07-01

Guanidinoacetate methyltransferase deficiency is an autosomal recessively inherited disorder of creatine biosynthesis. We report a new patient with guanidinoacetate methyltransferase deficiency and her >3-year treatment outcome. This is a 6-year-old girl who was diagnosed with guanidinoacetate methyltransferase deficiency at the age of 28 months. She presented with moderate global developmental delay, one afebrile seizure, and hypotonia between 6 and 18 months of life. She was treated with creatine and ornithine supplementation and a strict arginine-restricted diet for 42 months. Mutation analysis (compound heterozygous mutations, a known c.327G>A and a novel c.58dupT [p.Trp20LeufsX65]) and enzyme studies in primary fibroblasts confirmed the diagnosis. After 33 months of therapy, her cerebrospinal fluid guanidinoacetate level decreased from 47 to 5.3 times the normal level. Brain creatine by proton magnetic resonance spectroscopy increased by >75% but did not normalize in the basal ganglia and white matter after 3 years of therapy. Additional treatment with sodium benzoate for 17 months did not further improve plasma guanidinoacetate levels, which questions the relevance of this therapy. Treatment did not improve moderate intellectual disability or normalize guanidinoacetate accumulation in the central nervous system. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode

PubMed Central

Wagner, Tristan; Ermler, Ulrich; Shima, Seigo

2016-01-01

In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA–H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change. PMID:27324530

A novel BLAST-Based Relative Distance (BBRD) method can effectively group members of protein arginine methyltransferases and suggest their evolutionary relationship.

PubMed

Wang, Yi-Chun; Wang, Jing-Doo; Chen, Chin-Han; Chen, Yi-Wen; Li, Chuan

2015-03-01

We developed a novel BLAST-Based Relative Distance (BBRD) method by Pearson's correlation coefficient to avoid the problems of tedious multiple sequence alignment and complicated outgroup selection. We showed its application on reconstructing reliable phylogeny for nucleotide and protein sequences as exemplified by the fmr-1 gene and dihydrolipoamide dehydrogenase, respectively. We then used BBRD to resolve 124 protein arginine methyltransferases (PRMTs) that are homologues of nine mammalian PRMTs. The tree placed the uncharacterized PRMT9 with PRMT7 in the same clade, outside of all the Type I PRMTs including PRMT1 and its vertebrate paralogue PRMT8, PRMT3, PRMT6, PRMT2 and PRMT4. The PRMT7/9 branch then connects with the type II PRMT5. Some non-vertebrates contain different PRMTs without high sequence homology with the mammalian PRMTs. For example, in the case of Drosophila arginine methyltransferase (DART) and Trypanosoma brucei methyltransferases (TbPRMTs) in the analyses, the BBRD program grouped them with specific clades and thus suggested their evolutionary relationships. The BBRD method thus provided a great tool to construct a reliable tree for members of protein families through evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

How should immunomodulators be optimized when used as combination therapy with anti-tumor necrosis factor agents in the management of inflammatory bowel disease?

PubMed

Ward, Mark G; Irving, Peter M; Sparrow, Miles P

2015-10-28

In the last 15 years the management of inflammatory bowel disease has evolved greatly, largely through the increased use of immunomodulators and, especially, anti-tumor necrosis factor (anti-TNF) biologic agents. Within this time period, confidence in the use of anti-TNFs has increased, whilst, especially in recent years, the efficacy and safety of thiopurines has been questioned. Yet despite recent concerns regarding the risk: benefit profile of thiopurines, combination therapy with an immunomodulator and an anti-TNF has emerged as the recommended treatment strategy for the majority of patients with moderate-severe disease, especially those who are recently diagnosed. Concurrently, therapeutic drug monitoring has emerged as a means of optimizing the dosage of both immunomodulators and anti-TNFs. However the recommended therapeutic target levels for both drug classes were largely derived from studies of monotherapy with either agent, or studies underpowered to analyze outcomes in combination therapy patients. It has been assumed that these target levels are applicable to patients on combination therapy also, however there are few data to support this. Similarly, the timing and duration of treatment with immunomodulators when used in combination therapy remains unknown. Recent attention, including post hoc analyses of the pivotal registration trials, has focused on the optimization of anti-TNF agents, when used as either monotherapy or combination therapy. This review will instead focus on how best to optimize immunomodulators when used in combination therapy, including an evaluation of recent data addressing unanswered questions regarding the optimal timing, dosage and duration of immunomodulator therapy in combination therapy patients.

DHFR and MSH3 co-amplification in childhood acute lymphoblastic leukaemia, in vitro and in vivo.

PubMed

Matheson, Elizabeth C; Hogarth, Linda A; Case, Marian C; Irving, Julie A E; Hall, Andrew G

2007-06-01

The MSH3 and dihydrofolate reductase (DHFR) genes, located on chromosome 5, share a common promoter but are divergently transcribed. Dysregulation of the mismatch repair (MMR) pathway has been found to occur in cell line models due to co-amplification of MSH3 as a coincident effect of DHFR amplification, acquired as a mechanism generating resistance to methotrexate (MTX). The increased levels of MSH3 perturbed MutSalpha function resulting in hypermutability and increased resistance to thiopurines, drugs whose cytotoxic effects are triggered by MutSalpha. The relevance of this phenomenon in clinical samples is unknown but is extremely pertinent in childhood acute lymphoblastic leukaemia (ALL) in which children are exposed for prolonged periods to both MTX and thiopurines such that a single amplification event involving both the DHFR and the MSH3 genes may cause chemotherapeutic resistance to both agents. Thus, we have generated a leukaemic cell line (PreB697) and a normal human lymphoblastoid cell line (TK6) that are resistant to a pharmacologically relevant dose of MTX and show that while increased DHFR levels result in MTX resistance, the associated increased levels of MSH3 are insufficient to perturb MutSalpha functionality, in terms of MMR capacity or 6-thioguanine sensitivity. In addition, we show that although low-level DHFR amplification occurs alone in a significant number of samples, both at disease onset and relapse, co-amplification of both MSH3 and DHFR is rarely found in primary ALL samples, even after prolonged MTX therapy and is not at a sufficiently high level to perturb MMR function.

Collagenous colitis: Requirement for high-dose budesonide as maintenance treatment.

PubMed

Fernandez-Bañares, Fernando; Piqueras, Marta; Guagnozzi, Danila; Robles, Virginia; Ruiz-Cerulla, Alexandra; Casanova, María José; Gisbert, Javier P; Busquets, David; Arguedas, Yolanda; Pérez-Aisa, Angeles; Fernández-Salazar, Luis; Lucendo, Alfredo J

2017-09-01

Controlled studies show high efficacy of budesonide in inducing short-term clinical remission in collagenous colitis (CC), but relapses are common after its withdrawal. To evaluate the need for high-dose budesonide (≥6mg/d) to maintain clinical remission in CC. Analysis of a multicentre retrospective cohort of 75 patients with CC (62.3±1.5years; 85% women) treated with budesonide in a clinical practice setting between 2013 and 2015. Frequency of budesonide (9mg/d) refractoriness and safety, and the need for high-dose budesonide to maintain clinical remission, were evaluated. Drugs used as budesonide-sparing, including azathioprine and mercaptopurine, were recorded. Logistic regression analysis was performed to evaluate the risk factors associated with the need for high-dose budesonide (≥6mg/d) to maintain clinical remission. Budesonide induced clinical remission in 92% of patients, with good tolerance. Fourteen of 68 patients (21%; 95% CI, 13-32%) needed high-dose budesonide to maintain remission. Only intake of NSAIDs at diagnosis (OR, 8.6; 95% CI, 1.6-44) was associated with the need for high-dose budesonide in the multivariate analysis. with thiopurines was effective in 5 out of 6 patients (83%; 95% CI, 44-97%), allowing for withdrawal from or a dose decrease of budesonide. One fifth of CC patients, especially those with NSAID intake at diagnosis, require high-dose budesonide (≥6mg/d) to maintain clinical remission. In this setting, thiopurines might be effective as budesonide-sparing drugs. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Azathioprine with Allopurinol: Lower Deoxythioguanosine in DNA and Transcriptome Changes Indicate Mechanistic Differences to Azathioprine Alone.

PubMed

Coulthard, Sally A; Berry, Phil; McGarrity, Sarah; McLaughlin, Simon; Ansari, Azhar; Redfern, Christopher P F

2017-06-01

Use of azathioprine (AZA) for inflammatory bowel disease is limited by side effects or poor efficacy. Combining low-dose azathioprine with allopurinol (LDAA) bypasses side effects, improves efficacy, and may be appropriate as first-line therapy. We test the hypothesis that standard-dose azathioprine (AZA) and LDAA treatments work by similar mechanisms, using incorporation of the metabolite deoxythioguanosine into patient DNA, white-blood cell counts, and transcriptome analysis as biological markers of drug effect. DNA was extracted from peripheral whole-blood from patients with IBD treated with AZA or LDAA, and analyzed for DNA-incorporated deoxythioguanosine. Measurement of red-blood cell thiopurine metabolites was part of usual clinical practice, and pre- and on-treatment (12 wk) blood samples were used for transcriptome analysis. There were no differences in reduction of white-cell counts between the 2 treatment groups, but patients on LDAA had lower DNA-incorporated deoxythioguanosine than those on AZA; for both groups, incorporated deoxythioguanosine was lower in patients on thiopurines for 24 weeks or more (maintenance of remission) compared to patients treated for less than 24 weeks (achievement of remission). Patients on LDAA had higher levels of red-blood cell thioguanine nucleotides than those on AZA, but there was no correlation between these or their methylated metabolites, and incorporated deoxythioguanosine. Transcriptome analysis suggested down-regulation of immune responses consistent with effective immunosuppression in patients receiving LDAA, with evidence for different mechanisms of action between the 2 therapies. LDAA is biologically effective despite lower deoxythioguanosine incorporation into DNA, and has different mechanisms of action compared to standard-dose azathioprine.

Plant isoflavone and isoflavanone O-methyltransferase genes

DOEpatents

Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

2014-08-19

The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

Chromosomal 16S Ribosomal RNA Methyltransferase RmtE1 in Escherichia coli Sequence Type 448

PubMed Central

Li, Bin; Pacey, Marissa P.

2017-01-01

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone. PMID:28418308

Host Methyltransferases and Demethylases: Potential New Epigenetic Targets for HIV Cure Strategies and Beyond.

PubMed

Boehm, Daniela; Ott, Melanie

2017-11-01

A successful HIV cure strategy may require reversing HIV latency to purge hidden viral reservoirs or enhancing HIV latency to permanently silence HIV transcription. Epigenetic modifying agents show promise as antilatency therapeutics in vitro and ex vivo, but also affect other steps in the viral life cycle. In this review, we summarize what we know about cellular DNA and protein methyltransferases (PMTs) as well as demethylases involved in HIV infection. We describe the biology and function of DNA methyltransferases, and their controversial role in HIV infection. We further explain the biology of PMTs and their effects on lysine and arginine methylation of histone and nonhistone proteins. We end with a focus on protein demethylases, their unique modes of action and their emerging influence on HIV infection. An outlook on the use of methylation-modifying agents in investigational HIV cure strategies is provided.

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

PubMed

Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

2016-10-04

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G., E-mail: clarke@mbi.ucla.edu

2010-01-22

Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These resultsmore » are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

PubMed Central

Gopal, J; Yebra, M J; Bhagwat, A S

1994-01-01

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework. Images PMID:7971279

Development of Carbocyanine Dyes for PRMT Inhibition and Imaging

PubMed Central

Sinha, Sarmistha Halder; Owens, Eric A.; Feng, You; Yang, Yutao; Xie, Yan; Tu, Yaping; Henary, Maged; Zheng, Yujun George

2014-01-01

Summary Protein arginine methylation regulates multiple biological processes. Deregulation of protein arginine methyltransferase (PRMT) activities has been observed in many disease phenotypes. Small molecule probes that target PRMTs with strong affinity and selectivity can be used as valuable tools to dissect biological mechanisms of arginine methylation and establish the role of PRMT proteins in a disease process. In this work, we report synthesis and evaluation of a class of carbocyanine compounds containing indolium, benz[e]indolium or benz[c,d]indolium heterocyclic moieties that bind to the predominant arginine methyltransferase PRMT1 and inhibit its methyltransferase activity at low micromolar potencies. In particular, the developed molecules have long wavelength colorimetric and fluorometric photoactivities, which can be used for optical and near-infrared fluorescence imaging in cells or biological tissues. Together, these new chemical probes have potential application in PRMT studies both as enzyme inhibitors and as fluorescent dyes for microscope imaging. PMID:22749641

Absence of a direct role of phospholipid methylation in stimulus-secretion coupling and control of adenylate cyclase in guinea-pig and rat parotid gland.

PubMed Central

Padel, U; Unger, C; Söling, H D

1982-01-01

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland. PMID:6186246

The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers

PubMed Central

Russler-Germain, David A.; Spencer, David H.; Young, Margaret A.; Lamprecht, Tamara L.; Miller, Christopher A.; Fulton, Robert; Meyer, Matthew R.; Erdmann-Gilmore, Petra; Townsend, R. Reid; Wilson, Richard K.; Ley, Timothy J.

2014-01-01

Summary Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ~30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ~80% compared to the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity but co-expression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes. PMID:24656771

The role of protein methyltransferases as potential novel therapeutic targets in squamous cell carcinoma of the head and neck.

PubMed

Saloura, Vassiliki; Vougiouklakis, Theodore; Sievers, Cem; Burkitt, Kyunghee; Nakamura, Yusuke; Hager, Gordon; van Waes, Carter

2018-06-01

Squamous cell carcinoma of the head and neck is a lethal disease with suboptimal survival outcomes and standard therapies with significant comorbidities. Whole exome sequencing data recently revealed an abundance of genetic and expression alterations in a family of enzymes known as protein methyltransferases in a variety of cancer types, including squamous cell carcinoma of the head and neck. These enzymes are mostly known for their chromatin-modifying functions through methylation of various histone substrates, though evidence supports their function also through methylation of non-histone substrates. This review summarizes the current knowledge on the function of protein methyltransferases in squamous cell carcinoma of the head and neck and highlights their promising potential as the next generation of therapeutic targets in this disease. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

PubMed Central

McClelland, M; Nelson, M; Raschke, E

1994-01-01

Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

PubMed

Züst, Roland; Dong, Hongping; Li, Xiao-Feng; Chang, David C; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R; Ellis, Esther M; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Heterologous expression of 2-methylisoborneol / 2 methylenebornane biosynthesis genes in Escherichia coli yields novel C11-terpenes

PubMed Central

Wortmann, Hannah; Dickschat, Jeroen S.; Schrader, Jens

2018-01-01

The structural diversity of terpenoids is limited by the isoprene rule which states that all primary terpene synthase products derive from methyl-branched building blocks with five carbon atoms. With this study we discover a broad spectrum of novel terpenoids with eleven carbon atoms as byproducts of bacterial 2-methylisoborneol or 2-methylenebornane synthases. Both enzymes use 2-methyl-GPP as substrate, which is synthesized from GPP by the action of a methyltransferase. We used E. coli strains that heterologously produce different C11-terpene synthases together with the GPP methyltransferase and the mevalonate pathway enzymes. With this de novo approach, 35 different C11-terpenes could be produced. In addition to eleven known compounds, it was possible to detect 24 novel C11-terpenes which have not yet been described as terpene synthase products. Four of them, 3,4-dimethylcumene, 2-methylborneol and the two diastereomers of 2-methylcitronellol could be identified. Furthermore, we showed that an E. coli strain expressing the GPP-methyltransferase can produce the C16-terpene 6-methylfarnesol which indicates the condensation of 2-methyl-GPP and IPP to 6-methyl-FPP by the E. coli FPP-synthase. Our study demonstrates the broad range of unusual terpenes accessible by expression of GPP-methyltransferases and C11-terpene synthases in E. coli and provides an extended mechanism for C11-terpene synthases. PMID:29672609

Structure and expression of dna methyltransferase genes from apomictic and sexual Boechera species.

PubMed

Taşkin, Kemal Melik; Özbilen, Aslıhan; Sezer, Fatih; Hürkan, Kaan; Güneş, Şebnem

2017-04-01

In this study, we determined the structure of DNA methyltransferase (DNMT) genes in apomict and sexual Boechera species and investigated the expression levels during seed development. Protein and DNA sequences of diploid sexual Boechera stricta DNMT genes obtained from Phytozome 10.3 were used to identify the homologues in apomicts, Boechera holboellii and Boechera divaricarpa. Geneious R8 software was used to map the short-paired reads library of B. holboellii whole genome or B. divaricarpa transcriptome reads to the reference gene sequences. We determined three DNMT genes; for Boechera spp. METHYLTRANSFERASE1 (MET1), CHROMOMETHYLASE 3 (CMT3) and DOMAINS REARRANGED METHYLTRANSFERASE 1/2 (DRM2). We examined the structure of these genes with bioinformatic tools and compared with other DNMT genes in plants. We also examined the levels of expression in silique tissues after fertilization by semi-quantitative PCR. The structure of DNMT proteins in apomict and sexual Boechera species share common features. However, the expression levels of DNMT genes were different in apomict and sexual Boechera species. We found that DRM2 was upregulated in apomictic Boechera species after fertilization. Phylogenetic trees showed that three genes are conserved among green algae, monocotyledons and dicotyledons. Our results indicated a deregulation of DNA methylation machinery during seed development in apomicts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1*

PubMed Central

Lee, Byron H.; Yegnasubramanian, Srinivasan; Lin, Xiaohui; Nelson, William G.

2007-01-01

CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycyti-dine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer. PMID:16230360

Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

PubMed Central

Chang, David C.; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R.; Ellis, Esther M.; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response. PMID:23935499

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

PubMed

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Site-specific bioalkylation of rapamycin by the RapM 16-O-methyltransferase† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc00164a

PubMed Central

Law, Brian J. C.; Struck, Anna-Winona; Bennett, Matthew R.; Wilkinson, Barrie

2015-01-01

The methylation of natural products by S-adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors in vitro and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple S-ethyl or S-allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches. PMID:29403635

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houtz, Robert, L.

This project focused on a molecular and biochemical characterization of the protein methyltransferases responsible for methylation of the LS and SS in Rubisco, and the associated functional consequences accompanying these modifications. Our results provided some of the most informative structural and mechanistic understandings of SET domain protein methyltransferases. These results also positioned us to provide the first unambiguous assignment of the kinetic reaction mechanism for SET-domain protein methyltransferases, and to design and engineer an alternative substrate for Rubisco LSMT, enabling substrate specificity and functional significance studies. We demonstrated that the minimal substrate recognized by Rubisco LSMT is free lysine asmore » well as monomethyllysine, an observation corroborated both by structural analyses as well as enzymatic activity and subsequent product distribution analyses. Ternary complexes between Rubisco LSMT and free lysine compared to complexes with monomethyllysine demonstrated that the structural basis for multiple methyl group additions is a consequence of hydrogen-bond driven spatial shifts in the amino group of Lys-14, which maintains the direct in-line geometry necessary for SN2 nucleophilic attack. The structural observations are also consistent with the previous proposal that the multiplicity of methyl group additions takes place through a processive mechanism, with successive methyl group additions to an enzyme protein complex which does not disassociate prior to the formation of trimethyllysine. This mechanism has important implications, since the regulation of gene expression by SET domain histone methyltransferases is not only dependent on site-specific lysine methylation, but also the degree of methylation. We examined the kinetic reaction mechanism for three different types of SET domain protein methyltransferases, each under conditions supporting mono-, di-, or trimethyllysine formation corroborated by product analyses. Additionally, the tight initial binding of Rubisco LSMT to Rubisco also allowed us to design a novel immobilized complex between Rubisco and Rubisco LSMT, which allowed for an unambiguous demonstration of the requirement for trimethyllysine formation prior to disassociation of the Rubisco LSMT:Rubisco complex, and therefore proof of the processive mechanism for methyl group transfer. These kinetic studies also demonstrated that an important factor has been overlooked in all kinetic analyses of SET domain protein methyltransferases reported to date. This factor is the influence of the low turnover number for SET domain protein methyltransferases and how, relative to the time-frame of kinetic enzyme assays, this can generate changes in kinetic profiles shifting reciprocal plot patterns from random/ordered bi-bi to the real kinetic reaction mechanism plots of ping-pong. Although the ternary complexes of Rubisco LSMT with S-Adenosylhomocysteine and lysine and monomethyllysine were informative in regard to reaction mechanism, they were not helpful in identifying the mechanism used by Rubisco LSMT for determining substrate specificity. We were unsuccessful at obtaining ternary complexes of Rubisco LSMT with bound synthetic polypeptide substrates, as has been reported for several histone methyltransferases. However, we were able to model a polypeptide sequence corresponding to the N-terminal region of the LS of Rubisco into the apparent substrate binding cleft in Rubisco LSMT. Knowledge of the determinants of polypeptide substrate specificity are important for identifying possible alternate substrates, as well as the possibility of generating more desirable substrates amenable to site-directed mutagenesis experiments unlike Rubisco. We determined that Rubisco LSMT is capable of methylating synthetic polypeptide mimics of the N-terminal region of the LS, both free as well as conjugated to keyhole limpet hemacyanin, but with considerable less efficiency than intact holoenzyme.« less

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages. PMID:25187538

Structural and Functional Analysis of the Pyocyanin Biosynthetic Protein PhzM from Pseudomonas aeruginosa†‡

PubMed Central

Parsons, James F.; Greenhagen, Bryan T.; Shi, Katherine; Calabrese, Kelly; Robinson, Howard; Ladner, Jane E.

2008-01-01

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state. Pyocyanin is also a chemically interesting compound due to its unusual oxidation-reduction activity. Phenazine-1-carboxylic acid, the precursor to the bioactive phenazines, is synthesized from chorismic acid by enzymes encoded in a seven-gene cistron in Pseudomonas aeruginosa and in other Pseudomonads. Phenzine-1-carboxylic acid is believed to be converted to pyocyanin by the sequential actions of the putative S-adenosylmethionine dependent N-methyltransferase PhzM and the putative flavin-dependent hydroxylase PhzS. Here we report the 1.8 Å crystal structure of PhzM solved by single anomalous dispersion. Unlike many methyltransferases, PhzM is a dimer in solution. The 36 kDa PhzM polypeptide folds into three domains. The C-terminal domain exhibits the α/β-hydrolase fold typical of small molecule methyltransferases. Two smaller N-terminal domains form much of the dimer interface. Structural alignments with known methyltransferases show that PhzM is most similar to the plant O-methyltransferases that are characterized by an unusual intertwined dimer interface. The structure of PhzM contains no ligands and the active site is open and solvent exposed when compared to structures of similar enzymes. In vitro experiments using purified PhzM alone demonstrate that it has little or no ability to methylate phenzine-1-carboxylic acid. However, when the putative hydroxylase PhzS is included, pyocyanin is readily produced. This observation suggests that a mechanism has evolved in P. aeruginosa that ensures efficient production of pyocyanin by preventing the formation and release of an unstable and potentially deleterious intermediate. PMID:17253782

Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

PubMed

Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

2014-05-13

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20

PubMed Central

Yao, Li; Yu, Lin-Lu; Zhang, Jun-Jie; Xie, Xiang-Ting; Tao, Qing; Yan, Xin; Hong, Qing; Qiu, Ji-Guo

2016-01-01

ABSTRACT Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt. A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. IMPORTANCE Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops. PMID:27422839

Noncompetitive Inhibition of Indolethylamine-N-methyltransferase by N,N-Dimethyltryptamine and N,N-Dimethylaminopropyltryptamine

PubMed Central

2015-01-01

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis–Menten and Lineweaver–Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N′,N′-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14) identified an N-terminal helix–loop–helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were −6.34 and −7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT. PMID:24730580

Estrogen-DNA Adducts as Novel Biomarkers for Ovarian Cancer Risk and for Use in Prevention

DTIC Science & Technology

2013-03-01

genes for four selected estrogen-metabolizing enzymes : cytochrome P450 (CYP)1A1 (I462V), CYP1B1 (V432L),catechol-O-methyltransferase (COMT) (V158M...homozygous for the catechol-O-methyltransferase allele and the cytochrome P450 1B1 high activity allele had significantly increased DNA adduct ratios and... enzyme polymorphisms to serve as biomarkers to screen for ovarian cancer . Task 1. Obtain approval of the protocol from the OCRP Human Research

Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro▿

PubMed Central

Shin, Sung Jae; Collins, Michael T.

2008-01-01

The in vitro susceptibility of human- and bovine-origin Mycobacterium paratuberculosis to the thioupurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) was established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn's disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP, even at high concentrations (≥64.0 μg/ml). Among rapidly growing mycobacteria, Mycobacterium phlei was susceptible to 6-MP and AZA whereas Mycobacterium smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-aminosalycilic acid [mesalamine]) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin, used as control drugs, were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6-MP and ciprofloxacin resulted in significantly higher CFU than use of ciprofloxacin alone. These data may partially explain the paradoxical response of Crohn's disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs, i.e., they do not worsen with anti-inflammatory treatment as would be expected with a microbiological etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn's disease: AZA drugs may confound interpretation of data on therapeutic responses for both antibiotic-treated and control groups. PMID:18070971

The indirect antioxidant sulforaphane protects against thiopurine-mediated photooxidative stress

PubMed Central

Benedict, Andrea L.; Knatko, Elena V.; Dinkova-Kostova, Albena T.

2012-01-01

Long-term treatment with thiopurines, such as the widely used anticancer, immunosuppressive and anti-inflammatory agent azathioprine, combined with exposure to ultraviolet (UV) radiation is associated with increased oxidative stress, hyperphotosensitivity and high risk for development of aggressive squamous cell carcinomas of the skin. Sulforaphane, an isothiocyanate derived from broccoli, is a potent inducer of endogenous cellular defenses regulated by transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), including cytoprotective enzymes and glutathione, which in turn act as efficient indirect and direct antioxidants that have long-lasting effects. Treatment with 6-thioguanine, a surrogate for azathioprine, leads to profound sensitization to oxidative stress and glutathione depletion upon exposure to UVA radiation, the damaging effects of which are primarily mediated by generation of reactive oxygen species. The degree of sensitization is greater for irradiation exposures spanning the absorption spectrum of 6-thioguanine, and is dependent on the length of treatment and the level of guanine substitution with 6-thioguanine, suggesting that the 6-thioguanine that is incorporated in genomic DNA is largely responsible for this sensitization. Sulforaphane provides protection against UVA, but not UVB, radiation without affecting the levels of 6-thioguanine incorporation into DNA. The protective effect is lost under conditions of Nrf2 deficiency, implying that it is due to induction of Nrf2-dependent cytoprotective proteins, and that this strategy could provide protection against any potentially photosensitizing drugs that generate electrophilic or reactive oxygen species. Thus, our findings support the development of Nrf2 activators as protectors against drug-mediated photooxidative stress and encourage future clinical trials in populations at high risk for cutaneous photodamage and photocarcinogenesis. PMID:22983983

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.

PubMed

Yuan, Bifeng; Wang, Yinsheng

2008-08-29

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

Albumin as a prognostic marker for ulcerative colitis

PubMed Central

Khan, Nabeel; Patel, Dhruvan; Shah, Yash; Trivedi, Chinmay; Yang, Yu-Xiao

2017-01-01

AIM To evaluate the role of albumin at the time of ulcerative colitis (UC) diagnosis in predicting the clinical course of disease. METHODS Nationwide cohort of patients with newly diagnosed UC in the Veterans Affairs health care system was identified and divided into two categories: hypoalbuminemia (i.e., ≤ 3.5 gm/dL) or normal albumin levels (i.e., > 3.5 gm/dL) at the time of UC diagnosis. The exposure of interest was presence of hypoalbuminemia defined as albumin level ≤ 3.5 g/dL at the time of UC diagnosis. Patients were then followed over time to identify the use of ≥ 2 courses of corticosteroids (CS), thiopurines, anti-TNF medications and requirement of colectomy for UC management. RESULTS The eligible study cohort included 802 patients, but 92 (11.4%) patients did not have their albumin levels checked at the time of UC diagnosis, and they were excluded. A total of 710 patients, who had albumin levels checked at time of UC diagnosis, were included in our study. Amongst them, 536 patients had a normal albumin level and 174 patients had hypoalbuminemia. Patients with hypoalbuminemia at diagnosis had a higher likelihood of ≥ 2 courses of CS use (adjusted HR = 1.7, 95%CI: 1.3-2.3), higher likelihood of thiopurine or anti- TNF use (adjusted HR = 1.72, 95%CI: 1.23-2.40) than patients with normal albumin level at diagnosis. There was a trend of higher likelihood of colectomy in hypoalbuminemic patients, but it was not statistically significant (Adjusted HR = 1.7, 95%CI: 0.90-3.25). CONCLUSION Hypoalbuminemia at disease diagnosis can serve as a prognostic marker to predict the clinical course of UC at the time of diagnosis. PMID:29259376

The Toronto Consensus Statements for the Management of Inflammatory Bowel Disease in Pregnancy.

PubMed

Nguyen, Geoffrey C; Seow, Cynthia H; Maxwell, Cynthia; Huang, Vivian; Leung, Yvette; Jones, Jennifer; Leontiadis, Grigorios I; Tse, Frances; Mahadevan, Uma; van der Woude, C Janneke

2016-03-01

The management of inflammatory bowel disease (IBD) poses a particular challenge during pregnancy because the health of both the mother and the fetus must be considered. A systematic literature search identified studies on the management of IBD during pregnancy. The quality of evidence and strength of recommendations were rated using the Grading of Recommendation Assessment, Development and Evaluation (GRADE) approach. Consensus was reached on 29 of the 30 recommendations considered. Preconception counseling and access to specialist care are paramount in optimizing disease management. In general, women on 5-ASA, thiopurine, or anti-tumor necrosis factor (TNF) monotherapy for maintenance should continue therapy throughout pregnancy. Discontinuation of anti-TNF therapy or switching from combination therapy to monotherapy may be considered in very select low-risk patients. Women who have a mild to moderate disease flare while on optimized 5-ASA or thiopurine therapy should be managed with systemic corticosteroid or anti-TNF therapy, and those with a corticosteroid-resistant flare should start anti-TNF therapy. Endoscopy or urgent surgery should not be delayed during pregnancy if indicated. Decisions regarding cesarean delivery should be based on obstetric considerations and not the diagnosis of IBD alone, with the exception of women with active perianal Crohn's disease. With the exception of methotrexate, the use of medications for IBD should not influence the decision to breast-feed and vice versa. Live vaccinations are not recommended within the first 6 months of life in the offspring of women who were on anti-TNF therapy during pregnancy. Optimal management of IBD before and during pregnancy is essential to achieving favorable maternal and neonatal outcomes. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Extracolonic Cancer in Inflammatory Bowel Disease: Data from the GETECCU Eneida Registry.

PubMed

Chaparro, María; Ramas, M; Benítez, J M; López-García, A; Juan, A; Guardiola, J; Mínguez, M; Calvet, X; Márquez, L; Fernández Salazar, L I; Bujanda, L; García, C; Zabana, Y; Lorente, R; Barrio, J; Hinojosa, E; Iborra, M; Cajal, M Domínguez; Van Domselaar, M; García-Sepulcre, M F; Gomollón, F; Piqueras, M; Alcaín, G; García-Sánchez, V; Panés, J; Domènech, E; García-Esquinas, E; Rodríguez-Artalejo, F; Gisbert, J P

2017-07-01

The objective of this study was (a) To know the prevalence and distribution of extracolonic cancer (EC) in patients with inflammatory bowel disease (IBD); (b) To estimate the incidence rate of EC; (c) To evaluate the association between EC and treatment with immunosuppressants and anti-tumor necrosis factor (TNF) agents. This was an observational cohort study. IBD and inclusion in the ENEIDA Project (a prospectively maintained registry) from GETECCU. Patients with EC before the diagnosis of IBD, lack of relevant data for this study, and previous treatment with immunosuppressants other than corticosteroids, thiopurines, methotrexate, or anti-TNF agents. The Kaplan-Meier method was used to evaluate the impact of several variables on the risk of EC, and any differences between survival curves were evaluated using the log-rank test. Stepwise multivariate Cox regression analysis was used to investigate factors potentially associated with the development of EC, including drugs for the treatment of IBD, during follow-up. A total of 11,011 patients met the inclusion criteria and were followed for a median of 98 months. Forty-eight percent of patients (5,303) had been exposed to immunosuppressants or anti-TNF drugs, 45.8% had been exposed to thiopurines, 4.7% to methotrexate, and 21.6% to anti-TNF drugs. The prevalence of EC was 3.6%. In the multivariate analysis, age (HR=1.05, 95% CI=1.04-1.06) and having smoked (hazards ratio (HR)=1.47, 95% confidence interval (CI)=1.10-1.80) were the only variables associated with a higher risk of EC. Neither immunosuppressants nor anti-TNF drugs seem to increase the risk of EC. Older age and smoking were associated with a higher prevalence of EC.

The risk of lymphoma and immunomodulators in patients with inflammatory bowel diseases: results from a population-based cohort in Eastern Europe.

PubMed

Lakatos, Peter L; Lovasz, Barbara D; David, Gyula; Pandur, Tunde; Erdelyi, Zsuzsanna; Mester, Gabor; Balogh, Mihaly; Szipocs, Istvan; Molnar, Csaba; Komaromi, Erzsebet; Golovics, Petra A; Vegh, Zsuzsanna; Mandel, Michael; Horvath, Agnes; Szathmari, Miklos; Kiss, Lajos S; Lakatos, Laszlo

2013-06-01

Prior studies suggest a small but significantly increased risk of lymphoma in adults with inflammatory bowel disease (IBD), especially in patients treated with thiopurines. No data was available from Eastern Europe. The aim of this study was to analyze the incidence of lymphomas as related to drug exposure, in a population-based Veszprem province database, which included incident cases diagnosed between January 1, 1977 and December 31, 2008. Data from 1420 incident patients were analyzed (UC: 914, age at diagnosis: 36.5 years; CD: 506, age at diagnosis: 28.5.5 years). Both in- and outpatient records were collected and comprehensively reviewed. The rate of lymphoma was calculated as patient-years of exposure per medication class, of medications utilized in IBD. Of the 1420 patients, we identified three patients who developed lymphoma (one CLL, two low-grade B-cell NHL including one rectal case), during 19,293 patient-years of follow-up (median follow-up: 13 years). All three patients were male. None had received azathioprine or biologicals. The absolute incidence rate of lymphoma was 1.55 per 10,000 patient-years, with 3 cases observed vs. 2.18 expected, with a standardized incidence ratio (SIR) of 1.37 (95% confidence interval [CI]: 0.44-4.26). No cases have been exposed to either azathioprine or biologicals. The overall risk of lymphoma in IBD was not increased; only three cases were seen in this population-based incident cohort over a 30-year period. An association with thiopurine exposure was not found. Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

[Exclusive enteral nutrition continues to be first line therapy for pediatric Crohn's disease in the era of biologics].

PubMed

Navas-López, V M; Blasco-Alonso, J; Lacasa Maseri, S; Girón Fernández-Crehuet, F; Serrano Nieto, M J; Vicioso Recio, M I; Sierra Salinas, C

2015-07-01

Exclusive enteral nutrition (EEN) has been to be more effective than corticosteroids in achieving mucosal healing without their side effects. To determine the efficacy of EEN in terms of inducing clinical remission in newly diagnosed CD children and to study the efficacy of this therapeutic approach in improving the degree of intestinal mucosa inflammation. The medical records of patients with newly diagnosed Crohn's disease treated with EEN were reviewed retrospectively. The degree of mucosal inflammation was assessed by fecal calprotectin (FC). Remission was defined as a PCDAI<10. Forty patients (24 males) were included, the age at diagnosis was 11.6 ± 3.6 years. Of the 34 patients who completed the EEN period, 32 (94% per-protocol analysis) achieved clinical remission. This percentage fell to 80% in the intention-to-treat analysis. The compliance rate was 95%. Duration of EEN was 6.42 weeks (IQR 6.0-8.14). FC was significantly higher in patients with moderate and severe disease. Median baseline FC levels (680 μg/g) decreased significantly to 218 μg/g (P<0.0001) after EEN. We found a statistically significant correlation between FC and PCDAI (rho=0.727; P<0.0001). Early use of thiopurines (< 8 weeks) versus subsequent use was not associated with improved outcomes during the follow-up. EEN administered for 6-8 weeks is effective for inducing clinical remission and decreasing the degree of mucosal inflammation. We did not find differences in terms of maintenance of remission in patients treated early with thiopurines. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier España, S.L.U. All rights reserved.

Pneumocystis jirovecii Pneumonia in Pediatric Inflammatory Bowel Disease: A Case Report and Literature Review

PubMed Central

Lawrence, Sally J.; Sadarangani, Manish; Jacobson, Kevan

2017-01-01

Immunosuppressive therapy is a known risk factor for opportunistic infections. We report the first case of severe Pneumocystis jirovecii infection requiring intensive care in a pediatric patient with inflammatory bowel disease (IBD). The literature was reviewed and there were 92 reported cases of Pneumocystis pneumonia (PCP) in patients with IBD. Most sources were case reports and there was likely reporting bias toward patients receiving immunomodulators, anti-tumor necrosis factor (anti-TNF) therapy, and those who died. Overall, 56% of patients were males and 58% had Crohn’s disease. The median age was 45 years (interquartile range 30–68, range 8–78) and 86% of patients were lymphopenic. The case-fatality rate was 23%. Corticosteroids were used as IBD treatment in 88% of patients who subsequently developed PCP, 42% received thiopurines, 44% used anti-TNF therapy, and 15% received either cyclosporine or tacrolimus. Rates of mono, dual, triple, and quadruple immunosuppression therapy were 35, 35, 29, and 2%, respectively. This report highlights the importance of considering PCP in immunosuppressed lymphopenic pediatric IBD patients who present with unusual symptoms. Moreover, it should give gastroenterologists the impetus to limit immunosuppressive therapy to its minimal effective dose and consider options such as exclusive enteral nutrition wherever possible. Although there is no place for global PCP prophylaxis in IBD given the low incidence, in an era when there is increasing use of biologic agents with combination immunosuppressive therapy, the risk-benefit profile of PCP chemoprophylaxis should be revisited in selected cohorts such as patients on triple immunosuppression with corticosteroids, thiopurines, and a biological agent or calcineurin inhibitor, especially in lymphopenic individuals. PMID:28791279

Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2.

PubMed

Eggert, Erik; Hillig, Roman C; Koehr, Silke; Stöckigt, Detlef; Weiske, Jörg; Barak, Naomi; Mowat, Jeffrey; Brumby, Thomas; Christ, Clara D; Ter Laak, Antonius; Lang, Tina; Fernandez-Montalvan, Amaury E; Badock, Volker; Weinmann, Hilmar; Hartung, Ingo V; Barsyte-Lovejoy, Dalia; Szewczyk, Magdalena; Kennedy, Steven; Li, Fengling; Vedadi, Masoud; Brown, Peter J; Santhakumar, Vijayaratnam; Arrowsmith, Cheryl H; Stellfeld, Timo; Stresemann, Carlo

2016-05-26

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

The inhibition of the mammalian DNA methyltransferase 3a (Dnmt3a) by dietary black tea and coffee polyphenols

PubMed Central

2011-01-01

Background Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean. Results We studied the inhibition of DNA methyltransferase 3a by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance. Conclusions Since Dnnmt3a has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols. PMID:21510884

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

PubMed

Moore, M H; Gulbis, J M; Dodson, E J; Demple, B; Moody, P C

1994-04-01

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Synthesis and evaluation of carbocyanine dyes as PRMT inhibitors and imaging agents.

PubMed

Sinha, Sarmistha Halder; Owens, Eric A; Feng, You; Yang, Yutao; Xie, Yan; Tu, Yaping; Henary, Maged; Zheng, Yujun George

2012-08-01

Protein arginine methylation regulates multiple biological processes. Deregulation of protein arginine methyltransferase (PRMT) activities has been observed in many disease phenotypes. Small molecule probes that target PRMTs with strong affinity and selectivity can be used as valuable tools to dissect biological mechanisms of arginine methylation and establish the role of PRMT proteins in a disease process. In this work, we report synthesis and evaluation of a class of carbocyanine compounds containing indolium, benz[e]indolium or benz[c,d]indolium heterocyclic moieties that bind to the predominant arginine methyltransferase PRMT1 and inhibit its methyltransferase activity at low micromolar potencies. In particular, the developed molecules have long wavelength colorimetric and fluorometric photoactivities, which can be used for optical and near-infrared fluorescence imaging in cells or biological tissues. Together, these new chemical probes have potential application in PRMT studies both as enzyme inhibitors and as fluorescent dyes for microscope imaging. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Structure and function of the Zika virus full-length NS5 protein

DOE PAGES

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; ...

2017-03-27

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapetaniou, Evangelia G.; Kotsifaki, Dina; Providaki, Mary

2007-01-01

The DNA methyltransferase M.BseCI from B. stearothermophilus was crystallized as a complex with its cognate DNA. Crystals belong to space group P6 and diffract to 2.5 Å resolution at a synchrotron source. The DNA methyltransferase M.BseCI from Bacillus stearothermophilus (EC 2.1.1.72), a 579-amino-acid enzyme, methylates the N6 atom of the 3′ adenine in the sequence 5′-ATCGAT-3′. M.BseCI was crystallized in complex with its cognate DNA. The crystals were found to belong to the hexagonal space group P6, with unit-cell parameters a = b = 87.0, c = 156.1 Å, β = 120.0° and one molecule in the asymmetric unit. Twomore » complete data sets were collected at wavelengths of 1.1 and 2.0 Å to 2.5 and 2.8 Å resolution, respectively, using synchrotron radiation at 100 K.« less

Recruitment of DNA methyltransferase I to DNA repair sites.

PubMed

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M Cristina; Leonhardt, Heinrich

2005-06-21

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

PubMed Central

Zhong, Xuehua; Hale, Christopher J.; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A.; Henderson, Ian R.; Wohlschlegel, James A.; Jacobsen, Steven E.

2015-01-01

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine.

PubMed

Lee, Jin-Hyung; Cook, Jeffry R; Yang, Zhi-Hong; Mirochnitchenko, Olga; Gunderson, Samuel I; Felix, Arthur M; Herth, Nicole; Hoffmann, Ralf; Pestka, Sidney

2005-02-04

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

PubMed Central

Lukinavičius, Gražvydas; Lapinaitė, Audronė; Urbanavičiūtė, Giedrė; Gerasimaitė, Rūta; Klimašauskas, Saulius

2012-01-01

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA. PMID:23042683

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

PubMed

Kawamura, Takuya; Anraku, Ryou; Hasegawa, Takahiro; Tomikawa, Chie; Hori, Hiroyuki

2014-12-23

We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

PubMed Central

Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

2015-01-01

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

Cancer.gov

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

Two New Plant-Like Pathways Link Hemoglobin Degradation to Lipid Biogenesis in Falciparum Malaria: Novel Targets for Anti-Malarial Chemotherapy

DTIC Science & Technology

2005-03-01

when the compound was added at 50/uM (Appendix VI, B). Furthermore, clofibric acid , an inhibitor of PtdEtn methylransferases (19), had no effect on Pfpmt...reduced when the compound was added at 50 AM (Fig. 5B). Furthermore, clofibric acid , an inhibitor of PtdEtn B 35 methyltransferases (34), had no...Fig. 5B). Furthermore, clofibric acid , an takDs .Shnmn .Jnsn .Coday n .U~a o inhibitor of PtdEtn methyltransferases, had no effect on Pfpmt helpful

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn; Dong, Mingyue; Li, Ning

Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo andmore » maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA methyltransferase gene with conserved activity in both prokaryotic and eukaryotic non-host species.« less

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

PubMed Central

Motorin, Y; Grosjean, H

1999-01-01

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w. PMID:10445884

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

PubMed

Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)*

PubMed Central

Małecki, Jędrzej; Dahl, Helge-André; Moen, Anders; Davydova, Erna; Falnes, Pål Ø.

2016-01-01

Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the β-subunit of electron transfer flavoprotein (ETFβ). Interestingly, putative METTL20 orthologues are found in a subset of α-proteobacteria, including Agrobacterium tumefaciens. Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETFβ and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETFβ on Lys-193 and Lys-196 both in vitro and in vivo. ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETFβ. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven β-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETFβ and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETFβ as the first lysine methylation event occurring in both bacteria and humans. PMID:26929405

7-Methylxanthine methyltransferase of coffee plants. Gene isolation and enzymatic properties.

PubMed

Ogawa, M; Herai, Y; Koizumi, N; Kusano, T; Sano, H

2001-03-16

Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N. However, controversy exists as to the number and properties of the methyltransferases involved. Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants. Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves. Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT. It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring. K(m) values were estimated to be 50 and 12 microM for 7-methylxanthine and S-adenosyl-l-methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine.

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

PubMed Central

Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

2013-01-01

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis.

PubMed

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana.

PubMed

Hong, Sunghyun; Song, Hae-Ryong; Lutz, Kerry; Kerstetter, Randall A; Michael, Todd P; McClung, C Robertson

2010-12-07

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.

Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity.

PubMed

Aapola, Ulla; Liiv, Ingrid; Peterson, Pärt

2002-08-15

DNMT3L is a regulator of imprint establishment of normally methylated maternal genomic sequences. DNMT3L shows high similarity to the de novo DNA methyltransferases, DNMT3A and DNMT3B, however, the amino acid residues needed for DNA cytosine methyltransferase activity have been lost from the DNMT3L protein sequence. Apart from methyltransferase activity, Dnmt3a and Dnmt3b serve as transcriptional repressors associating with histone deacetylase (HDAC) activity. Here we show that DNMT3L can also repress transcription by binding directly to HDAC1 protein. We have identified the PHD-like zinc finger of the ATRX domain as a main repression motif of DNMT3L, through which DNMT3L recruits the HDAC activity needed for transcriptional silencing. Furthermore, we show that DNMT3L protein contains an active nuclear localisation signal at amino acids 156-159. These results describe DNMT3L as a co-repressor protein and suggest that a transcriptionally repressed chromatin organisation through HDAC activity is needed for establishment of genomic imprints.

Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yan; Li, Fengling; Babault, Nicolas

G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including 4 (MS0124) and 18more » (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme.« less

Computational fishing of new DNA methyltransferase inhibitors from natural products.

PubMed

Maldonado-Rojas, Wilson; Olivero-Verbel, Jesus; Marrero-Ponce, Yovani

2015-07-01

DNA methyltransferase inhibitors (DNMTis) have become an alternative for cancer therapies. However, only two DNMTis have been approved as anticancer drugs, although with some restrictions. Natural products (NPs) are a promising source of drugs. In order to find NPs with novel chemotypes as DNMTis, 47 compounds with known activity against these enzymes were used to build a LDA-based QSAR model for active/inactive molecules (93% accuracy) based on molecular descriptors. This classifier was employed to identify potential DNMTis on 800 NPs from NatProd Collection. 447 selected compounds were docked on two human DNA methyltransferase (DNMT) structures (PDB codes: 3SWR and 2QRV) using AutoDock Vina and Surflex-Dock, prioritizing according to their score values, contact patterns at 4 Å and molecular diversity. Six consensus NPs were identified as virtual hits against DNMTs, including 9,10-dihydro-12-hydroxygambogic, phloridzin, 2',4'-dihydroxychalcone 4'-glucoside, daunorubicin, pyrromycin and centaurein. This method is an innovative computational strategy for identifying DNMTis, useful in the identification of potent and selective anticancer drugs. Copyright © 2015 Elsevier Inc. All rights reserved.

Recent advances in targeting protein arginine methyltransferase enzymes in cancer therapy.

PubMed

Smith, Emily; Zhou, Wei; Shindiapina, Polina; Sif, Said; Li, Chenglong; Baiocchi, Robert A

2018-05-21

Exploration in the field of epigenetics has revealed the diverse roles of the protein arginine methyltransferase (PRMT) family of proteins in multiple disease states. These findings have led to the development of specific inhibitors and discovery of several new classes of drugs with potential to treat both benign and malignant conditions. Areas covered: We provide an overview on the role of PRMT enzymes in healthy and malignant cells, highlighting the role of arginine methylation in specific pathways relevant to cancer pathogenesis. Additionally, we describe structure and catalytic activity of PRMT and discuss the mechanisms of action of novel small molecule inhibitors of specific members of the arginine methyltransferase family. Expert opinion: As the field of PRMT biology advances, it's becoming clear that this class of enzymes is highly relevant to maintaining normal physiologic processes as well and disease pathogenesis. We discuss the potential impact of PRMT inhibitors as a broad class of drugs, including the pleiotropic effects, off target effects the need for more detailed PRMT-centric interactomes, and finally, the potential for targeting this class of enzymes in clinical development of experimental therapeutics for cancer.

Arginine methyltransferase inhibitor 1 inhibits gastric cancer by downregulating eIF4E and targeting PRMT5.

PubMed

Zhang, Baolai; Zhang, Su; Zhu, Lijuan; Chen, Xue; Zhao, Yunfeng; Chao, Li; Zhou, Juanping; Wang, Xing; Zhang, Xinyang; Ma, Nengqian

2017-12-01

Arginine methylation is carried out by protein arginine methyltransferase (PRMTs) family. Arginine methyltransferase inhibitor 1 (AMI-1) is mainly used to inhibit type I PRMT activity in vitro. However, the effects of AMI-1 on type II PRMT5 activity and gastric cancer (GC) remain unclear. In this study, we provided the first evidence that AMI-1 significantly inhibited GC cell proliferation and migration while induced GC cell apoptosis, and reduced the expression of PRMT5, eukaryotic translation initiation factor 4E (eIF4E), symmetric dimethylation of histone 3 (H3R8me2s) and histone 4 (H4R3me2s). In addition, AMI-1 inhibited tumor growth, downregulated eIF4E, H4R3me2s and H3R8me2s expression in mice xenografts model of GC. Collectively, our results suggest that AMI-1 inhibits GC by downregulating eIF4E and targeting type II PRMT5. Copyright © 2017 Elsevier Inc. All rights reserved.

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity.

PubMed

Takahashi, Yuta; Daitoku, Hiroaki; Yokoyama, Atsuko; Nakayama, Kimihiro; Kim, Jun-Dal; Fukamizu, Akiyoshi

2011-04-01

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.

Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.

Cloning, expression, purification and preliminary X-­ray crystallographic analysis of mouse protein arginine methyltransferase 7

PubMed Central

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. PMID:24419624

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis

PubMed Central

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen. PMID:28493897

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

PubMed Central

Hong, Sunghyun; Lutz, Kerry; Kerstetter, Randall A.; Michael, Todd P.; McClung, C. Robertson

2010-01-01

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general. PMID:21097700

Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation.

PubMed

Małecki, Jędrzej; Jakobsson, Magnus E; Ho, Angela Y Y; Moen, Anders; Rustan, Arild C; Falnes, Pål Ø

2017-10-27

Lysine methylation is an important and much-studied posttranslational modification of nuclear and cytosolic proteins but is present also in mitochondria. However, the responsible mitochondrial lysine-specific methyltransferases (KMTs) remain largely elusive. Here, we investigated METTL12, a mitochondrial human S -adenosylmethionine (AdoMet)-dependent methyltransferase and found it to methylate a single protein in mitochondrial extracts, identified as citrate synthase (CS). Using several in vitro and in vivo approaches, we demonstrated that METTL12 methylates CS on Lys-395, which is localized in the CS active site. Interestingly, the METTL12-mediated methylation inhibited CS activity and was blocked by the CS substrate oxaloacetate. Moreover, METTL12 was strongly inhibited by the reaction product S -adenosylhomocysteine (AdoHcy). In summary, we have uncovered a novel human mitochondrial KMT that introduces a methyl modification into a metabolic enzyme and whose activity can be modulated by metabolic cues. Based on the established naming nomenclature for similar enzymes, we suggest that METTL12 be renamed CS-KMT (gene name CSKMT ). © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

DOEpatents

Houtz, Robert L.

2001-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Soon Goo; Alpert, Tara D.; Jez, Joseph M.

2012-07-17

Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0 {angstrom} resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC{sub 50} values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.

A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Nan; Ferrer, Jean-Luc; Moon, Hong S

2012-01-01

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested formore » methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.« less

LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2

DOE PAGES

Nguyen, Hannah; Allali-Hassani, Abdellah; Antonysamy, Stephen; ...

2015-03-30

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex withmore » LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. As a result, these findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.« less

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

PubMed

Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

2016-04-04

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Dietary intake of S-(α-carboxybutyl)-DL-homocysteine induces hyperhomocysteinemia in rats

PubMed Central

Strakova, Jana; Williams, Kelly T.; Gupta, Sapna; Schalinske, Kevin L.; Kruger, Warren D.; Rozen, Rima; Jiracek, Jiri; Li, Lucas; Garrow, Timothy A.

2010-01-01

Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(α-carboxybutyl)-DL-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed for the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an L-amino acid-defined diet either containing or devoid of CBHcy (5 mg/meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine β-synthase activity and immunodetectable protein decreased (56 and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats and S-adenosylhomocysteine levels did not change. Further, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology and plasma markers of liver damage. We conclude that CBHcy mediated BHMT inhibition causes an elevation in total plasma homocysteine that is not normalized by the folate-dependent conversion of homocysteine to methionine. Further, metabolic changes caused by BHMT inhibition affect cystathionine β-synthase and glycine N-methyltransferase activities, which further deteriorate plasma homocysteine levels. PMID:20797482

Gene expression differences in the methionine remethylation and transsulphuration pathways under methionine restriction and recovery with D,L-methionine or D,L-HMTBA in meat-type chickens.

PubMed

Aggrey, S E; González-Cerón, F; Rekaya, R; Mercier, Y

2018-02-01

This study examined the molecular mechanisms of methionine pathways in meat-type chickens where birds were provided with a diet deficient in methionine from 3 to 5 weeks of age. The birds on the deficient diet were then provided with a diet supplemented with either D,L-methionine or D,L-HMTBA from 5 to 7 weeks. The diet of the control birds was supplemented with L-methionine from hatch till 7 weeks of age. We studied the mRNA expression of methionine adenosyltransferase 1, alpha, methionine adenosyltransferase 1, beta, 5-methyltetrahydrofolate-homocysteine methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase, betaine-homocysteine S-methyltransferase, glycine N-methyltransferase, S-adenosyl-L-homocysteine hydrolase and cystathionine beta synthase genes in the liver, duodenum, Pectoralis (P.) major and the gastrocnemius muscle at 5 and 7 weeks. Feeding a diet deficient in dietary methionine affected body composition. Birds that were fed a methionine-deficient diet expressed genes that indicated that remethylation occurred via the one-carbon pathway in the liver and duodenum; however, in the P. major and the gastrocnemius muscles, gene expression levels suggested that homocysteine received methyl from both folate and betaine for remethylation. Birds who were switched from a methionine deficiency diet to one supplemented with either D,L-methionine or D,L-HMTBA showed a downregulation of all the genes studied in the liver. However, depending on the tissue or methionine form, either folate or betaine was elicited for remethylation. Thus, mRNA expressions show that genes in the remethylation and transsulphuration pathways were regulated according to tissue need, and there were some differences in the methionine form. © 2017 Blackwell Verlag GmbH.

LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Hannah; Allali-Hassani, Abdellah; Antonysamy, Stephen

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex withmore » LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. As a result, these findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.« less

Alternation of histone and DNA methylation in human atherosclerotic carotid plaques.

PubMed

Greißel, A; Culmes, M; Napieralski, R; Wagner, E; Gebhard, H; Schmitt, M; Zimmermann, A; Eckstein, H-H; Zernecke, A; Pelisek, J

2015-08-01

Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.

Molecular cloning and characterization of tetrahydroprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis in opium poppy.

PubMed

Liscombe, David K; Facchini, Peter J

2007-05-18

S-Adenosyl-l-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K(m) values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 microm, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

PubMed Central

Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1997-01-01

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

PubMed

Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

1997-05-13

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity.

PubMed

Gursoy-Yuzugullu, Ozge; Carman, Chelsea; Serafim, Rodolfo Bortolozo; Myronakis, Marios; Valente, Valeria; Price, Brendan D

2017-04-11

Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.

Use of allopurinol in children with acute lymphoblastic leukemia to reduce skewed thiopurine metabolism.

PubMed

Brackett, Julienne; Schafer, Eric S; Leung, Daniel H; Bernhardt, M Brooke

2014-06-01

Mercaptopurine (6-MP), a critical component of acute lymphoblastic leukemia (ALL) therapy, is metabolized to 6-thioguanine (6-TGN) which is responsible for its anti-leukemic effect, and to 6-methylmercaptopurine nucleotides (6-MMPN/6-MMP) which can be hepatotoxic. Some patients preferentially metabolize 6-MP to 6-MMPN which may increase the risk of liver injury, reduce serum levels of 6-TGN and potentially increase the risk of relapse. The addition of allopurinol to oral 6-MP has been shown to optimize metabolism towards 6-TGN in patients with inflammatory bowel disease (IBD); however, this use has not been reported in patients undergoing treatment for ALL. © 2013 Wiley Periodicals, Inc.

Hodgkin's Lymphoma in Crohn's Disease Treated with Infliximab

PubMed Central

Carvalho, Diana; Russo, Pedro; Bernardes, Carlos; Saiote, Joana; Ramos, Gonçalo; Mascarenhas, Luís; Borges, Nuno; Ramos, Jaime

2017-01-01

Introduction Lymphoproliferative disorders, particularly non-Hodgkin's and Hodgkin's lymphomas, are rare in patients with inflammatory bowel diseases. The use of thiopurines and infection by Epstein-Barr virus are well-known cofactors that can raise its prevalence. Other risk factors such as disease activity and biological treatment are the subject of discussion, without enough data in the literature to confirm a potential association. Methods We report a case of Hodgkin's lymphoma in a patient who had been treated with azathioprine and was on long-term monotherapy with infliximab. Conclusions We stress the importance of recognizing the possible occurrence of a lymphoproliferative disorder in association with anti-tumor necrosis factor-α therapy PMID:29255769

Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction. | Office of Cancer Genomics

Cancer.gov

Epigenetic modulators play critical roles in reprogramming of cellular functions, emerging as a new class of promising therapeutic targets. Nuclear receptor binding SET domain protein 3 (NSD3) is a member of the lysine methyltransferase family. Interestingly, the short isoform of NSD3 without the methyltransferase fragment, NSD3S, exhibits oncogenic activity in a wide range of cancers. We recently showed that NSD3S interacts with MYC, a central regulator of tumorigenesis, suggesting a mechanism by which NSD3S regulates cell proliferation through engaging MYC.

No association between catechol-O-methyltransferase polymorphisms and neurotic disorders among mainland Chinese university students.

PubMed

Kou, Changgui; Meng, Xiangfei; Xie, Bing; Shi, Jieping; Yu, Qiong; Yu, Yaqin; D'Arcy, Carl

2012-07-30

This study investigates the genetic association between catechol-O-methyltransferase (COMT) gene polymorphisms and neurotic disorders. Data were derived from a case-control association study of 255 undergraduates affected by neurotic disorders and 269 matched healthy undergraduate controls. The polymorphisms of eight tag single nucleotide polymorphisms (SNPs) on the COMT gene were tested using polymerase chain reaction (PCR)-based Ligase Detection Reaction (PCR-LDR). The eight tag SNPs on the COMT gene assessed were not associated with neurotic disorders. Our finding suggests that the COMT gene may not be a susceptibility gene for neurotic disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

QM/MM MD and Free Energy Simulation Study of Methyl Transfer Processes Catalyzed by PKMTs and PRMTs.

PubMed

Chu, Yuzhuo; Guo, Hong

2015-09-01

Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here, we review the results of quantum mechanics/molecular mechanics molecular dynamics and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes.

QM/MM MD and free energy simulation study of methyl transfer processes catalyzed by PKMTs and PRMTs.

PubMed

Chu, Yuzhuo; Guo, Hong

2015-01-16

Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here we review the results of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes.

An easy-to-perform photometric assay for methyltransferase activity measurements.

PubMed

Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

2013-01-01

Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

PubMed Central

Yang, Xin; Yang, Ying; Sun, Bao-Fa; Chen, Yu-Sheng; Xu, Jia-Wei; Lai, Wei-Yi; Li, Ang; Wang, Xing; Bhattarai, Devi Prasad; Xiao, Wen; Sun, Hui-Ying; Zhu, Qin; Ma, Hai-Li; Adhikari, Samir; Sun, Min; Hao, Ya-Juan; Zhang, Bing; Huang, Chun-Min; Huang, Niu; Jiang, Gui-Bin; Zhao, Yong-Liang; Wang, Hai-Lin; Sun, Ying-Pu; Yang, Yun-Gui

2017-01-01

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation. PMID:28418038

Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

DOE PAGES

Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; ...

2015-03-12

Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties ofmore » PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.« less

The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olsson, Ida; Berrez, Jean-Marc; Leipus, Arunas

2007-05-15

Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experimentsmore » with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2{delta} mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation.« less

The story of protein arginine methylation: characterization, regulation, and function.

PubMed

Peng, Chao; Wong, Catherine Cl

2017-02-01

Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery. Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases. Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garg, Aprajita; Lukk, Tiit; Kumar, Vidya

Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties ofmore » PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.« less

Biosynthesis of diphthamide in the yeast Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, J.Y.C.

1985-01-01

Inactivation of EF-2 by diphtheria toxin requires the presence of a posttranslationally synthesized amino acid residue, diphthamide. The present work was undertaken to study the biosynthetic mechanism of diphthamide synthesis in the yeast Saccharomyces cerevisiae in order to gain better understanding of the biological roles of this unique amino acid residue. Thirty-one haploid ADP-ribosylation-negative mutants, comprising 5 complementation groups, were obtained. One of these mutants contains a toxin-resistant form of EF-2 which can be converted to a toxin-sensitive form through the methylation reaction catalyzed by a S-AdoMet:EF-2 methyltransferase enzyme which is present in other yeast strains. The (/sup 3/He)methylated residuemore » in the EF-2 modified by the methyltransferase in the presence of S-Ado-L-(/sup 3/H-methyl)-Met has been analyzed chromatographically following both acid and enzymatic hydrolysis. At the conclusion of the reaction, all of the radiolabel was recovered as diphthine (the unamidated form of diphthamide). The authors conclude that the S-AdoMet:EF-2-methyltransferase is specific for the addition of at least the last two of the three methyl groups present in diphthine.« less

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

PubMed

Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

2014-05-06

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

Dimethylsulfoniopropionate biosynthesis in a diatom Thalassiosira pseudonana: Identification of a gene encoding MTHB-methyltransferase.

PubMed

Kageyama, Hakuto; Tanaka, Yoshito; Shibata, Ayumi; Waditee-Sirisattha, Rungaroon; Takabe, Teruhiro

2018-05-01

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira. Copyright © 2018 Elsevier Inc. All rights reserved.

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of largemore » heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.« less

Purification and Characterization of S-Adenosyl-l-Methionine: Desoxyhemigossypol-6-O-Methyltransferase from Cotton Plants. An Enzyme Capable of Methylating the Defense Terpenoids of Cotton1

PubMed Central

Liu, Jinggao; Benedict, Chauncey R.; Stipanovic, Robert D.; Bell, Alois A.

1999-01-01

Cotton contains a unique group of terpenoids including desoxyhemigossypol, hemigossypol, gossypol, hemigossypolone, and the heliocides that are part of the plant's defense system against pathogenic fungi and insects. Desoxyhemigossypol is a key intermediate in the biosynthesis of these compounds. We have isolated, purified, and characterized from cotton stele tissue infected with Verticillium dahliae a methyltransferase (S-adenosyl-l-Met: desoxyhemigossypol-6-O-methyltransferase) that specifically methylates the 6-position of desoxyhemigossypol to form desoxyhemigossypol-6-methyl ether with a Km value of 4.5 μm for desoxyhemigossypol and a Kcat/Km of 5.08 × 104 s−1 (mol/L)−1. The molecular mass of the native enzyme is 81.4 kD and is dissociated into two subunits of 41.2 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The enzymatic reaction does not require Mg+2 and is inhibited 98% with 10 mm p-chloromercuribenzoate. Desoxyhemigossypol-6-methyl ether leads to the biosynthesis of methylated hemigossypol, gossypol, hemigossypolone, and the heliocides, which lowers their effectiveness as phytoalexins and insecticides. PMID:10557251

Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

PubMed

Roy, A; Roy Chattopadhyay, N

2013-07-01

Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. Copyright © 2013. Published by Elsevier Ltd.

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA

PubMed Central

Ranaei-Siadat, Ehsan; Fabret, Céline; Seijo, Bili; Dardel, Frédéric; Grosjean, Henri; Nonin-Lecomte, Sylvie

2013-01-01

In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5′ and 3′ ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m5UψC. In tRNAs of gram-negative bacteria, formation of m5U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m5U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m5U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m5U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m5U in RNA was performed by NMR analysis. PMID:23603891

A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil1[C][W][OA

PubMed Central

Berim, Anna; Hyatt, David C.; Gang, David R.

2012-01-01

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 4′- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4′-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation. PMID:22923679

An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata

PubMed Central

Li, Jia; Li, Changfu; Gou, Junbo; Wang, Xin; Fan, Rongyan; Zhang, Yansheng

2016-01-01

The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4′-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4′-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4′-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo. PMID:27379141

An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata.

PubMed

Li, Jia; Li, Changfu; Gou, Junbo; Wang, Xin; Fan, Rongyan; Zhang, Yansheng

2016-01-01

The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4'-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4'-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4'-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo.

TERRA and the histone methyltransferase Dot1 cooperate to regulate senescence in budding yeast

PubMed Central

Wanat, Jennifer J.; Logsdon, Glennis A.; Driskill, Jordan H.; Deng, Zhong; Lieberman, Paul M.

2018-01-01

The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (tlc1Δ). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA in vivo and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of EXO1, TEL1, SAS2, and genes encoding RNase H enzymes. Further, it acts independently of the senescence delay provided by RAD52-dependent recombination. However, anti-TERRA delays senescence in a fashion epistatic to inactivation of the conserved histone methyltransferase Dot1. Dot1 associates with TERRA, and anti-TERRA disrupts this interaction in vitro and in vivo. Surprisingly, the anti-TERRA delay is independent of the C-terminal methyltransferase domain of Dot1 and instead requires only its N-terminus, which was previously found to facilitate release of telomeres from the nuclear periphery. Together, these data suggest that TERRA and Dot1 cooperate to drive senescence. PMID:29649255

Identification of novel inhibitors of DNA methylation by screening of a chemical library.

PubMed

Ceccaldi, Alexandre; Rajavelu, Arumugam; Ragozin, Sergey; Sénamaud-Beaufort, Catherine; Bashtrykov, Pavel; Testa, Noé; Dali-Ali, Hana; Maulay-Bailly, Christine; Amand, Séverine; Guianvarc'h, Dominique; Jeltsch, Albert; Arimondo, Paola B

2013-03-15

In order to discover new inhibitors of the DNA methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC50 of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC50 at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficiently reactivated YFP gene expression in a stable HEK293 cell line by promoter demethylation. Their efficacy was comparable to the DNMT inhibitor of reference 5-azacytidine.

Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

PubMed

Karner, Susanne; Shi, Shaojun; Fischer, Christine; Schaeffeler, Elke; Neurath, Markus F; Herrlinger, Klaus R; Hofmann, Ute; Schwab, Matthias

2010-04-01

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Incidence, management, and course of cancer in patients with inflammatory bowel disease.

PubMed

Algaba, Alicia; Guerra, Iván; Marín-Jiménez, Ignacio; Quintanilla, Elvira; López-Serrano, Pilar; García-Sánchez, María Concepción; Casis, Begoña; Taxonera, Carlos; Moral, Ignacio; Chaparro, María; Martín-Rodríguez, Daniel; Martín-Arranz, María Dolores; Manceñido, Noemí; Menchén, Luis; López-Sanromán, Antonio; Castaño, Ángel; Bermejo, Fernando

2015-04-01

Patients with inflammatory bowel disease [IBD] are at increased risk for developing some types of neoplasia. Our aims were to determin the risk for cancer in patients with IBD and to describe the relationship with immunosuppressive therapies and clinical management after tumor diagnosis. Retrospective, multicenter, observational, 5-year follow-up, cohort study. Relative risk [RR] of cancer in the IBD cohort and the background population, therapeutic strategies, and cancer evolution were analyzed. A total of 145 cancers were diagnosed in 133 of 9100 patients with IBD (global cumulative incidence 1.6% vs 2.4% in local population; RR = 0.67; 95% confidence interval [CI]: 0.57-0.78). Patients with IBD had a significantly increased RR of non-melanoma skin cancer [RR = 3.85; 2.53-5.80] and small bowel cancer [RR = 3.70; 1.23-11.13]. After cancer diagnosis, IBD treatment was maintained in 13 of 27 [48.1%] patients on thiopurines, in 2 of 3 on methotrexate [66.6%], none on anti-TNF-α monotherapy [n = 6] and 4 of 12 [33.3%] patients on combined therapy. Rate of death and cancer remission during follow-up did not differ [p > 0.05] between patients who maintained the treatment compared with patients who withdrew [5% vs 8% and 95% vs 74%, respectively]. An association between thiopurines [p = 0.20] or anti-TNF-α drugs [p = 0.77] and cancer was not found. Patients with IBD have an increased risk for non-melanoma skin cancer and small bowel cancer. Immunosuppresive therapy is not related to a higher overall risk for cancer or worse tumor evolution in patients who maintain these drugs after cancer diagnosis. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Gene set analysis of purine and pyrimidine antimetabolites cancer therapies.

PubMed

Fridley, Brooke L; Batzler, Anthony; Li, Liang; Li, Fang; Matimba, Alice; Jenkins, Gregory D; Ji, Yuan; Wang, Liewei; Weinshilboum, Richard M

2011-11-01

Responses to therapies, either with regard to toxicities or efficacy, are expected to involve complex relationships of gene products within the same molecular pathway or functional gene set. Therefore, pathways or gene sets, as opposed to single genes, may better reflect the true underlying biology and may be more appropriate units for analysis of pharmacogenomic studies. Application of such methods to pharmacogenomic studies may enable the detection of more subtle effects of multiple genes in the same pathway that may be missed by assessing each gene individually. A gene set analysis of 3821 gene sets is presented assessing the association between basal messenger RNA expression and drug cytotoxicity using ethnically defined human lymphoblastoid cell lines for two classes of drugs: pyrimidines [gemcitabine (dFdC) and arabinoside] and purines [6-thioguanine and 6-mercaptopurine]. The gene set nucleoside-diphosphatase activity was found to be significantly associated with both dFdC and arabinoside, whereas gene set γ-aminobutyric acid catabolic process was associated with dFdC and 6-thioguanine. These gene sets were significantly associated with the phenotype even after adjusting for multiple testing. In addition, five associated gene sets were found in common between the pyrimidines and two gene sets for the purines (3',5'-cyclic-AMP phosphodiesterase activity and γ-aminobutyric acid catabolic process) with a P value of less than 0.0001. Functional validation was attempted with four genes each in gene sets for thiopurine and pyrimidine antimetabolites. All four genes selected from the pyrimidine gene sets (PSME3, CANT1, ENTPD6, ADRM1) were validated, but only one (PDE4D) was validated for the thiopurine gene sets. In summary, results from the gene set analysis of pyrimidine and purine therapies, used often in the treatment of various cancers, provide novel insight into the relationship between genomic variation and drug response.

A thiopurine drug inhibits West Nile virus production in cell culture, but not in mice.

PubMed

Lim, Pei-Yin; Keating, Julie A; Hoover, Spencer; Striker, Rob; Bernard, Kristen A

2011-01-01

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.

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judge, Russell A.; Zhu, Haizhong; Upadhyay, Anup K.

SETD8 is a histone H4–K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

Effects of phenylethanolamine N-methyltransferase inhibitors on uptake and release of norepinephrine and dopamine from rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, N.Y.; Hower, J.A.; Borchardt, R.T.

1985-09-01

Inhibitors of phenylethanolamine N-methyltransferase (PNMT) and amphetamine were evaluated for their effects on the uptake of (TH)-norepinephrine (TH-NE) and the release of endogenous NE and dopamine (DA) from chopped rat brain tissues. Unlike amphetamine, all of PNMT inhibitors tested produced only slight inhibition of (TH)-NE uptake into chopped cerebral cortex. 2,3-Dichloro-alpha-methylbenzylamine (DCMB) and 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (SKF64139), but not 2-cyclooctyl-2-hydroxyethylamine (CONH) and 1-aminomethylcycloundecanol (CUNH) produced slight release of endogenous NE and DA from chopped hypothalami, but their effects were less pronounced than those produced by amphetamine.

Guanidinoacetate methyltransferase deficiency: the first inborn error of creatine metabolism in man.

PubMed Central

Stöckler, S.; Isbrandt, D.; Hanefeld, F.; Schmidt, B.; von Figura, K.

1996-01-01

In two children with an accumulation of guanidinoacetate in brain and a deficiency of creatine in blood, a severe deficiency of guanidinoacetate methyltransferase (GAMT) activity was detected in the liver. Two mutant GAMT alleles were identified that carried a single base substitution within a 5' splice site or a 13-nt insertion and gave rise to four mutant transcripts. Three of the transcripts encode truncated polypeptides that lack a residue known to be critical for catalytic activity of GAMT. Deficiency of GAMT is the first inborn error of creatine metabolism. It causes a severe developmental delay and extrapyramidal symptoms in early infancy and is treatable by oral substitution with creatine. Images Figure 2 PMID:8651275

Assay of 6-thioinosinic acid and 6-thioguanine nucleotides, active metabolites of 6-mercaptopurine, in human red blood cells.

PubMed

Lennard, L

1987-12-25

A highly sensitive reversed-phase high-performance liquid chromatographic assay, with ultraviolet detection, for 6-thioinosinic acid and the 6-thioguanine nucleotides (6TGNs) was developed. The 6TGNs are major red blood cell metabolites of the immunosuppressive agent azathioprine and the cytotoxic drugs 6-thioguanine and 6-mercaptopurine. The assay is based on the specific extraction, via phenyl mercury adduct formation, of the thiopurine released on acid hydrolysis of the thionucleotide metabolite. Red blood cell 6TGN concentrations in eighteen leukaemic children receiving chronic 6-mercaptopurine chemotherapy were measured and compared to a previously published spectrophotofluorometric assay. Linear regression analysis gave r = 0.991; P less than 0.001; y = 40 + 0.94x.

Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

PubMed

Byeon, Yeong; Back, Kyoungwhan

2016-08-01

Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-11-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed Central

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-01-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla. Images PMID:2903502