ABSTRACT
The objective of this study was to investigate the effect of 460 nm light-emitting diode (LED) on the inactivation of foodborne bacteria. Additionally, the change in the endogenous metabolic profile of LED illuminated cells was analyzed to understand the bacterial response to the LED illumination. Six different species of bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella Typhimurium) were illuminated with 460 nm LED to a maximum dose of 4080 J/cm2 at 4, 10 and 25 °C. Inactivation curves were modeled using Hom model. Metabolic profiling of the non-illuminated and illuminated cells was performed using a Liquid chromatography-mass spectrometry system. Results indicate that the 460 nm LED significantly (p < 0.05) reduced the populations of all six bacterial species. For example, the population of S. aureus reached below detection limit within 7 h. B. cereus was most resistant to photo-inactivation and exhibited about 3-log reduction in 9 h. Metabolic profiling of the illuminated cells indicated that several metabolites e.g. 11-deoxycortisol, actinonin, coformycin, tyramine, chitobiose etc. were regulated during LED illumination. These results elucidate the effectiveness of 460 nm LED against foodborne bacteria and hence, its suitability as a novel antimicrobial control method to ensure food safety.
Subject(s)
Bacteria/radiation effects , Food Safety/methods , Light , Metabolome , Microbial Viability/radiation effects , Bacteria/growth & development , Chromatography, Liquid , Coformycin/metabolism , Colony Count, Microbial , Escherichia coli O157/growth & development , Escherichia coli O157/radiation effects , Food Microbiology/methods , Food Preservation/methods , Hydroxamic Acids/metabolism , Limit of Detection , Listeria monocytogenes/growth & development , Listeria monocytogenes/radiation effects , Metabolome/radiation effects , Oxidative Stress , TemperatureABSTRACT
Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.
Subject(s)
Adenosine Deaminase/metabolism , Coformycin/analogs & derivatives , Coformycin/chemistry , Coformycin/metabolism , Malaria, Falciparum/enzymology , Plasmodium falciparum/enzymology , Adenosine Deaminase/chemistry , Animals , Antimalarials/chemistry , Antimalarials/metabolism , Antimalarials/pharmacology , Coformycin/pharmacology , Humans , Malaria, Falciparum/metabolism , Malaria, Falciparum/prevention & control , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Structure-Activity Relationship , Substrate SpecificityABSTRACT
AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Hypoglycemic Agents/pharmacology , Liver/metabolism , Mitochondria, Liver , Multienzyme Complexes/metabolism , Oxidative Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Ribonucleosides/pharmacology , AMP-Activated Protein Kinases , Aminoimidazole Carboxamide/pharmacology , Animals , Cells, Cultured , Coformycin/metabolism , Dose-Response Relationship, Drug , Electron Transport Complex I/physiology , Enzyme Activation , Enzyme Inhibitors/metabolism , Fructose/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Multienzyme Complexes/genetics , Oxygen/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Rats, WistarABSTRACT
Adenosine 5'-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5'-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6(2)22, with unit-cell parameters a = b = 131.325, c = 208.254 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.34 A resolution from a crystal in complex with coformycin 5'-phosphate and to 4.05 A resolution from a crystal of a mercury derivative.
Subject(s)
AMP Deaminase/chemistry , AMP Deaminase/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , Coformycin/metabolism , Macromolecular Substances/chemistry , Organophosphates/chemistry , Coformycin/chemistry , Crystallization , Crystallography, X-Ray , Data CollectionABSTRACT
Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermodynamically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are -4.7 +/- 0.8 kJ/mole-K and -1.2 +/- 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect. Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.
Subject(s)
Adenosine Deaminase/metabolism , Coformycin/metabolism , Protein Structure, Tertiary , Tubercidin/metabolism , Adenosine Deaminase/chemistry , Adenosine Deaminase Inhibitors , Animals , Binding Sites , Cattle , Coformycin/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Mathematics , Molecular Structure , Protein Binding , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/metabolism , Temperature , Thermodynamics , Tubercidin/chemistryABSTRACT
A series of N3-substituted coformycin aglycon analogues are described that inhibit adenosine 5'-monophosphate deaminase (AMPDA) or adenosine deaminase (ADA). The key steps involved in the preparation of these compounds are (1) treating the sodium salt of 6, 7-dihydroimidazo[4,5-d][1,3]diazepin-8(3H)-one (4) with an alkyl bromide or an alkyl mesylate to generate the N3-alkylated compound 5 and (2) reducing 5 with NaBH(4). Selective inhibition of AMPDA was realized when the N3-substituent contained a carboxylic acid moiety. For example, compound 7b which has a hexanoic acid side chain inhibited AMPDA with a K(i) = 4.2 microM and ADA with a K(i) = 280 microM. Substitution of large lipophilic groups alpha to the carboxylate provided a moderate potency increase with maintained selectivity as exemplified by the alpha-benzyl analogue 7j (AMPDA K(i) = 0.41 microM and ADA K(i) > 1000 microM). These compounds, as well as others described in this series of papers, are the first compounds suitable for testing whether selective inhibition of AMPDA can protect tissue from ischemic damage by increasing local adenosine concentrations at the site of injury and/or by minimizing adenylate loss.
Subject(s)
AMP Deaminase/antagonists & inhibitors , Coformycin/analogs & derivatives , Coformycin/chemical synthesis , Enzyme Inhibitors/chemical synthesis , AMP Deaminase/chemistry , Animals , Cattle , Cell Membrane/metabolism , Cells, Cultured , Coformycin/chemistry , Coformycin/metabolism , Endothelium/cytology , Endothelium/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Erythrocytes/metabolism , Ischemia/prevention & control , Liver/cytology , Magnetic Resonance Spectroscopy , Rabbits , Rats , Structure-Activity RelationshipABSTRACT
Arrhenius plots of the interactions of bovine adenosine deaminase (ADA) and of coformycin-inhibited ADA with adenosine are non-linear and reveal that coformycin significantly increases the activation energy for reaction only at temperatures well below the normal operating temperature of the enzyme (38.3 degrees C). This apparent enhanced affinity of the enzyme for the transition state analog at low temperature is confirmed from determinations of coformycin binding at 38.3 degrees C (KI = 5.3 x 10(-11) M) and at 21 degrees C (KI = 1.1 x 10(-11) M). It is suggested that these data are inconsistent with a model for general enzyme catalysis that requires an initial transition state complementary active site. Instead, it is suggested that an initial active site transition state complementarity is undesirable and the tendency of the enzyme to exist in this conformer at low temperatures is responsible for its inefficient interaction with adenosine substrate.
Subject(s)
Adenosine Deaminase/metabolism , Coformycin/metabolism , Adenosine/metabolism , Adenosine Deaminase Inhibitors , Animals , Binding Sites , Catalysis , Cattle , Coformycin/pharmacology , Cold Temperature , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Kinetics , Protein Binding , Spleen/enzymology , TemperatureABSTRACT
The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool.
Subject(s)
AMP Deaminase/metabolism , AMP Deaminase/antagonists & inhibitors , Adenosine Deaminase/metabolism , Adenosine Deaminase Inhibitors , Adenosine Triphosphate/metabolism , Animals , Cattle , Coformycin/analogs & derivatives , Coformycin/metabolism , Coformycin/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Herbicides/metabolism , Herbicides/pharmacology , In Vitro Techniques , Pisum sativum/drug effects , Pisum sativum/metabolism , RabbitsABSTRACT
Molecular dynamics and free energy simulations were performed to examine the binding of (8R)-deoxycoformycin and (8R)-coformycin to adenosine deaminase. The two inhibitors differ only at the 2' position of the sugar ring; the sugar moiety of conformycin is ribose, while it is deoxyribose for deoxycoformycin. The 100 ps molecular dynamics trajectories reveal that Asp 19 and His 17 interact strongly with the 5' hydroxyl group of the sugar moiety of both inhibitors and appear to play an important role in binding the sugar. The 2' and 3' groups of the sugars are near the protein-water interface and can be stabilized by either protein residues or water. The flexibility of the residues at the opening of the active site helps to explain the modest difference in binding of the two inhibitors and how substrates/inhibitors can enter an otherwise inaccessible binding site.
Subject(s)
Adenosine Deaminase Inhibitors , Coformycin/pharmacology , Pentostatin/pharmacology , Adenosine Deaminase/metabolism , Binding Sites , Coformycin/metabolism , Deoxyribose/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Models, Molecular , Molecular Conformation , Molecular Structure , Pentostatin/metabolism , Protein Binding , Ribose/metabolism , Thermodynamics , WaterABSTRACT
Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.
Subject(s)
Adenosine Deaminase/isolation & purification , Brain/enzymology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine Deaminase/metabolism , Adenosine Deaminase Inhibitors , Animals , Cattle , Coformycin/metabolism , Coformycin/pharmacology , Cytoplasm/enzymology , Enzyme Stability , In Vitro Techniques , Isoelectric Point , Kinetics , Molecular Weight , Pentostatin/metabolism , Pentostatin/pharmacology , TemperatureABSTRACT
Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.
Subject(s)
AMP Deaminase/metabolism , Nucleotide Deaminases/metabolism , Saccharomyces cerevisiae/enzymology , AMP Deaminase/antagonists & inhibitors , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Binding Sites , Binding, Competitive , Coformycin/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Phosphorus/metabolism , Protein Binding , Temperature , UltrafiltrationABSTRACT
The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.
Subject(s)
Leukemia, Myeloid, Acute/metabolism , Nucleosides/metabolism , Adenosine/metabolism , Adenosine Deaminase Inhibitors , Cell Differentiation/drug effects , Cell Line , Coformycin/analogs & derivatives , Coformycin/metabolism , Deoxyadenosines/metabolism , Dimethylformamide/pharmacology , Humans , Leukemia, Myeloid, Acute/pathology , Pentostatin , Purines/metabolism , Thioinosine/analogs & derivatives , Thioinosine/metabolismABSTRACT
Under conditions where 2'-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5'-phosphate in 15-20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of less than or equal to 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5'-phosphate in 20% overall yield. Coformycin-5'-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2'-deoxycoformycin-5'-phosphate, coformycin-5'-phosphate was a feeble substrate of snake venom 5'-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5'-AMP. With 5'-AMP analogues as substrate, the 5'-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values greater than 0.3 mM. The 5'-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki greater than 0.2 mM), but are potent inhibitors of adenylate deaminase (Ki less than or equal to 10(-9) M). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5'-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.
Subject(s)
Coformycin/metabolism , Nucleosides/metabolism , Nucleotides/metabolism , Ribonucleosides/metabolism , 5'-Nucleotidase , AMP Deaminase/metabolism , Adenosine Deaminase/metabolism , Chromatography, Thin Layer , Coformycin/analogs & derivatives , Magnetic Resonance Spectroscopy , Nucleotidases/analysis , Pentostatin , Phosphorylation , Purine-Nucleoside Phosphorylase/analysis , Snake Venoms/analysis , Spectrophotometry, UltravioletABSTRACT
Cerebral blood flow in the rat was monitored by a venous outflow technique with an extracorporeal circulation, which allows for the continuous recording of flow over periods of several hours. The adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (1.0-100 micrograms/kg) and deoxycoformycin (0.1-1 micrograms/kg) potentiated the reactive hyperemia elicited by a brief (24-s) anoxic challenge. Basal flow rate was unaltered by EHNA administration and slightly enhanced by deoxycoformycin. The results are consistent with the hypothesis that adenosine plays a significant role in cerebral vascular regulation and suggest that low doses of these deaminase inhibitors may be useful in the treatment of cerebral vascular insufficiency.
Subject(s)
Adenine/analogs & derivatives , Cerebrovascular Circulation/drug effects , Coformycin/pharmacology , Hyperemia/metabolism , Hypoxia, Brain/metabolism , Ribonucleosides/pharmacology , Adenine/metabolism , Adenine/pharmacology , Adenosine Deaminase Inhibitors , Animals , Brain/enzymology , Brain/metabolism , Coformycin/analogs & derivatives , Coformycin/metabolism , Male , Pentostatin , Rats , Rats, Inbred StrainsSubject(s)
Adenosine Deaminase/deficiency , Immunologic Deficiency Syndromes/enzymology , Nucleoside Deaminases/deficiency , Adenosine Deaminase/genetics , Adenosine Deaminase/isolation & purification , Adenosine Deaminase Inhibitors , Blood Transfusion , Cell Line , Coformycin/analogs & derivatives , Coformycin/metabolism , Coformycin/pharmacology , Humans , Immunologic Deficiency Syndromes/metabolism , Immunologic Deficiency Syndromes/therapy , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacology , Infant , Lymphocytes/enzymology , Lymphocytes/metabolism , Lymphoproliferative Disorders/drug therapy , Male , Mutation , Pentostatin , RNA, Messenger/metabolismABSTRACT
The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 X 10(-3) M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 microM when the extracellular concentration was 1 microM. At 10 microM extracellular concentration, the intracellular concentration was 3-4 microM. Although deoxycoformycin at very low concentrations (1 or 10 microM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase.
Subject(s)
Adenosine Deaminase Inhibitors , Coformycin/metabolism , DNA/metabolism , Nucleoside Deaminases/antagonists & inhibitors , Ribonucleosides/metabolism , T-Lymphocytes/metabolism , Adenosine Kinase/metabolism , Chromatography, High Pressure Liquid , Coformycin/analogs & derivatives , Deoxyadenine Nucleotides/metabolism , Deoxyadenosines/metabolism , Deoxycytidine Kinase/metabolism , Humans , Pentostatin , Phosphorylation , RNA/metabolism , Vidarabine/pharmacologyABSTRACT
Adenosine deaminase (adenosine aminohydrolase, EC3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.
Subject(s)
Adenosine Deaminase/blood , Erythrocytes/enzymology , Isoenzymes/blood , Nucleoside Deaminases/blood , Adenosine Deaminase Inhibitors , Catalysis , Coformycin/analogs & derivatives , Coformycin/metabolism , Humans , Hydrogen-Ion Concentration , Molecular WeightABSTRACT
A flow-limited physiologic mathematical model has been developed to describe the time course of 2'deoxycoformycin (2'dCF) concentrations in the plasma and tissues of mice following iv and ip doses. Urinary excretion is modeled as a linear involving filtration and secretion, since kidney clearance exceeded estimated glomerular filtration rate. Intracellular binding is described as the sum of linear nonspecific binding plus strong saturable binding to adenosine deaminase. Pharmacokinetic parameters are determined by a sequential optimization scheme in which each tissue is studied by means of a hybrid model. The model has been used to predict pharmacokinetic behaviour of 2'dCF in both normal and leukemic mice, and model simulations are compared with published data.
Subject(s)
Adenosine Deaminase Inhibitors , Coformycin/metabolism , Leukemia L1210/metabolism , Nucleoside Deaminases/antagonists & inhibitors , Ribonucleosides/metabolism , Adenosine Deaminase/metabolism , Animals , Coformycin/analogs & derivatives , Glomerular Filtration Rate , Kidney/metabolism , Kinetics , Mathematics , Mice , Mice, Inbred Strains , Models, Biological , Pentostatin , Tissue DistributionABSTRACT
2'-deoxycoformycin (2'-dCF) is a powerful inhibitor of adenosine deaminase (ADA), an enzyme found in high concentrations in lymphoid tissue. Although inactive in preclinical tumor models, 2'-dCF has shown clinical antitumor activity as a single agent in lymphoid malignancies. This drug has the added potential of being useful as a potentiator of other antitumor agents which are deactivated by ADA. It is also possible that the drug has potential as an immunosuppressive agent. Phase I studies are ongoing and phase II trials are planned to define the antitumor spectrum of this agent.
Subject(s)
Antineoplastic Agents , Coformycin/pharmacology , Ribonucleosides/pharmacology , Animals , Coformycin/analogs & derivatives , Coformycin/metabolism , Coformycin/therapeutic use , Coformycin/toxicity , Humans , Kinetics , Neoplasms, Experimental/drug therapy , PentostatinABSTRACT
2'-deoxycoformycin (2'-dCF; Pentostatin), a stoichiometric inhibitor of mammalian adenosine deaminase (ado deaminase), exhibits immunosuppressive and antilymphocytic activity in animal test systems. A clinical pharmacology/phase I study of 2'-dCF administered as a single agent has been completed (18 patients). Dose levels ranged from 0.1 mg/kg X 1 to 0.25 mg/kg/day X 5; ado deaminase and 2'-dCF were measured spectrophotometrically. Plasma decay curves were bi-exponential (alpha and beta t 1/2 values about 1 and 10 h respectively). Recovery of unchanged 2'-dCF from urine (48 h) was 32%--48% of the administered drug. Major toxic manifestations were lymphocytopenia (all patients) and urate nephropathy (1 patient, with subsequent patients in the series receiving allopurinol, 300 mg/day). Three partial responses were seen in seven patients with acute lymphocytic leukaemia receiving 0.25 mg 2'-dCF/kg/day X 5.