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Bioorganic & Medicinal Chemistry Sep 2015MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine...
MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied. Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are nanomolar inhibitors.
Topics: Adenine; Drug Discovery; Escherichia coli; Humans; Models, Molecular; Neoplasms; Plasmodium falciparum; Protein Binding; Pseudomonas aeruginosa; Purine-Nucleoside Phosphorylase; Pyrrolidines; Stereoisomerism
PubMed: 26260335
DOI: 10.1016/j.bmc.2015.07.059 -
Journal of Crohn's & Colitis Dec 2014The thiopurines are widely used in the treatment of inflammatory bowel disease, but are limited by poor dose-effect relationship. The objective was to assess the ability...
BACKGROUND AND AIMS
The thiopurines are widely used in the treatment of inflammatory bowel disease, but are limited by poor dose-effect relationship. The objective was to assess the ability of a novel assay, determining the mono-, di-, and triphosphates, of thioguanine as well as methylthioinosine as individual metabolites in erythrocytes, to predict clinical outcome compared to a routine assay, determining metabolites as sums.
METHODS
Samples from 79 patients with Crohn's disease or ulcerative colitis treated with azathioprine or mercaptopurine were analysed by both assays. Clinical status was determined by the Harvey-Bradshaw and Walmsley indices. The genotypes of thiopurine methyltransferase (TPMT) and inosine triphosphatase were determined.
RESULTS
TPMT wild-type patients with thioguanine nucleotide (TGN) levels below the cut-off level were more likely to have active disease when TGN was measured by the novel assay (p=0.02), and when thioguanosine triphosphate (TGTP) was measured separately (p=0.01). When TGN was measured by the routine assay the correlation was not evident (p=0.12). Neither TGN levels nor TGTP correlated to disease activity in TPMT deficient patients. Patients with methyl thioinosine nucleotide (meTIN) levels above 1500 pmol/8×10^8 RBCs were more likely to have active disease (p=0.07). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R(2)>0.88).
CONCLUSIONS
The novel TGN assay was better in predicting clinical outcome compared to the routine assay, while determination of TGTP had no clinical advantage and TGTP ratio was not correlated to disease activity.
Topics: Adolescent; Adult; Aged; Azathioprine; Cross-Sectional Studies; Drug Monitoring; Erythrocytes; Female; GTP Phosphohydrolases; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methylthioinosine; Middle Aged; Thioguanine; Thioinosine; Thionucleotides; Young Adult
PubMed: 25239576
DOI: 10.1016/j.crohns.2014.08.009 -
Journal of Pharmaceutical and... Sep 2014In the treatment of inflammatory bowel diseases, the use of azathioprine is increasing over the time. It has been demonstrated that the effectiveness of this therapy is...
In the treatment of inflammatory bowel diseases, the use of azathioprine is increasing over the time. It has been demonstrated that the effectiveness of this therapy is modulated by the metabolism of azathioprine, which is mainly exerted by both thiopurine methyl-transferase and inosine triphosphatase enzymes. Several studies reported chromatographic methods to determine the amount of its metabolites in erythrocytes, but there are not reported methods to dose them in peripheral blood mononuclear cells (PBMCs). The development of a method capable to quantify azathioprine nucleoside metabolites in this compartment could give better information on drug penetration and metabolism in the active site. In this work, we validated a new chromatographic method suitable for the monitoring of the two major biologically active ribonucleos(t)ide metabolites of azathioprine in PBMCs: 6-thioguanosine and 6-methyl-mercaptopurine riboside. After PBMCs extraction from blood through separation on density gradient, samples underwent a de-phosphorylation procedure with acid phosphatase (only one aliquot for each sample) and were then treated with a protein precipitation protocol in acetonitrile, followed by UPLC-tandem-mass spectrometry analysis. The calibration curve for each metabolite in PBMC fitted a least squares model (weighed 1/X) from 0.048 to 25ng (r(2)=0.998). Both accuracy and precision parameters fitted FDA guidelines. We tested this method by monitoring the concentrations of each metabolite in PBMC from eight inflammatory bowel diseases affected patients, receiving azathioprine maintenance therapy with optimal results.
Topics: Azathioprine; Chromatography, High Pressure Liquid; Guanosine; Humans; Inflammatory Bowel Diseases; Leukocytes, Mononuclear; Methylthioinosine; Tandem Mass Spectrometry; Thionucleosides
PubMed: 24960235
DOI: 10.1016/j.jpba.2014.05.040 -
Nucleosides, Nucleotides & Nucleic Acids 2014Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP... (Clinical Trial)
Clinical Trial
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.
Topics: Adolescent; Antineoplastic Agents; Child; Child, Preschool; Erythrocytes; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 24940700
DOI: 10.1080/15257770.2014.904519 -
Eukaryotic Cell May 2014The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside...
The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5'-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5'-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2'-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5'-methylthioinosine and similarly 5'-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.
Topics: Catalysis; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Kinetics; Protozoan Proteins; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Substrate Specificity; Toxoplasma
PubMed: 24585883
DOI: 10.1128/EC.00308-13 -
Marine Drugs Feb 2014A new actinomycete strain Micromonospora sp. K310 was isolated from Ghanaian mangrove river sediment. Spectroscopy-guided fractionation led to the isolation of two new... (Comparative Study)
Comparative Study
A new actinomycete strain Micromonospora sp. K310 was isolated from Ghanaian mangrove river sediment. Spectroscopy-guided fractionation led to the isolation of two new compounds from the fermentation culture. One of the compounds is butremycin (2) which is the (3-hydroxyl) derivative of the known Streptomyces metabolite ikarugamycin (1) and the other compound is a protonated aromatic tautomer of 5'-methylthioinosine (MTI) (3). Both new compounds were characterized by 1D, 2D NMR and MS data. Butremycin (2) displayed weak antibacterial activity against Gram-positive S. aureus ATCC 25923, the Gram-negative E. coli ATCC 25922 and a panel of clinical isolates of methicillin-resistant S. aureus (MRSA) strains while 3 did not show any antibacterial activity against these microbes.
Topics: Anti-Bacterial Agents; Escherichia coli; Fermentation; Geologic Sediments; Ghana; Lactams, Macrocyclic; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methicillin-Resistant Staphylococcus aureus; Methylthioinosine; Microbial Sensitivity Tests; Micromonospora; Rivers; Staphylococcus aureus
PubMed: 24534843
DOI: 10.3390/md12020999 -
PloS One 2014Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that...
Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.
Topics: Amino Acid Sequence; Catalytic Domain; Kinetics; Methylthioinosine; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Plasmodium falciparum; Purine-Nucleoside Phosphorylase; Pyrimidinones; Structure-Activity Relationship; Substrate Specificity
PubMed: 24416224
DOI: 10.1371/journal.pone.0084384 -
Biochemical and Biophysical Research... Jul 2013The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic...
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.
Topics: Antineoplastic Agents; Cell Line, Tumor; Humans; Mercaptopurine; Methyltransferases; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; RNA, Small Interfering; Thioguanine
PubMed: 23811272
DOI: 10.1016/j.bbrc.2013.06.067 -
Journal of Pharmaceutical and... Mar 2013A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs...
A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 10⁸ RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling.
Topics: Adult; Analytic Sample Preparation Methods; Antimetabolites, Antineoplastic; Azathioprine; Biotransformation; Chromatography, High Pressure Liquid; Drug Monitoring; Erythrocytes; Female; Humans; Indicators and Reagents; Male; Mercaptopurine; Nucleic Acid Synthesis Inhibitors; Pilot Projects; Potassium Permanganate; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purines; Quaternary Ammonium Compounds; Thioguanine; Thionucleotides
PubMed: 23261807
DOI: 10.1016/j.jpba.2012.11.027 -
Biochemistry Nov 2012Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl...
Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.
Topics: Adenosine Deaminase; Amino Acid Sequence; Coformycin; Crystallography, X-Ray; Deoxyadenosines; Humans; Ligases; Methylthioinosine; Models, Molecular; Molecular Sequence Data; Nucleoside Deaminases; Pseudomonas aeruginosa; Quorum Sensing; Sequence Alignment; Substrate Specificity; Thionucleosides
PubMed: 23050701
DOI: 10.1021/bi301062y