Inhibition of human purine nucleoside phosphorylase by acyclic nucleosides and nucleotides

In an effort to develop more potent inhibitors of human purine nucleoside phosphorylase (PNP) as immunosuppressive and cancer chemotherapeutic agents, the affinity of the erythrocytic enzyme for 30 acyclic nucleosides, nucleotides and related compounds was determined. Among the acyclonucleosides, 2'-nordeoxyguanosine [2'NDG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine] had a 3-fold greater affinity than acyclovir, and 8-amino-2'NDG was the best inhibitor with Ki = 2.6 X 10(-7) M. The ether moiety of the acyclovir and 2'NDG side-chains was not important for binding. Phosphorylated 2'NDG analogs appeared to act as multisubstrate analogs with optimal binding at low (1 mM) phosphate concentration. The 2'NDG mono- and triphosphates had higher affinities than those reported for the phosphorylated acyclovir derivatives but the diphosphate had a similar Ki value of 9 X 10(-9) M. Poor affinity, independent of phosphate concentration, was found for 9-(2-phosphonoethyl)guanine. The 3'-phosphate derivative of 8-(3-hydroxypropyl)-9-methylguanine inhibited with a Ki = 2 X 10(-5) M in 1 mM phosphate. The chemical syntheses of new analogs are described.     

Inhibition of liver alcohol dehydrogenase and ethanol metabolism by 3-substituted thiolane 1-oxides

3-Substituted thiolane 1-oxides (methyl, n-butyl, n-hexyl, and phenyl) were prepared and tested as inhibitors of horse, monkey, and rat liver alcohol dehydrogenases and of ethanol metabolism in rats. These compounds inhibit alcohol oxidation in an uncompetitive manner with respect to ethanol as a varied substrate. Lengthening the alkyl substituent increased the inhibitory potency because of tighter binding in the hydrophobic substrate binding pocket of the alcohol dehydrogenases. Thus, the 3-hexyl derivative was the most potent inhibitor of the purified rat liver alcohol dehydrogenase, with a Kii value of 0.13 microM. The 3-butyl derivative was the best inhibitor of ethanol metabolism in rats, with a Kii value of 11 mumol/kg. The acute toxicity in mice of the butyl derivative was 1.4 mmol/kg. Since high concentrations of alcohol do not prevent the inhibitory effects of these compounds, they may be particularly useful for preventing poisoning by methanol or ethylene glycol.     

C(2′)-substituted purine nucleoside analogs: Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides

Four C(2′)-substituted 2′-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine, 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine, 9-(2′-azido-2′-deoxy-β-D-ribofuranosyl)adenine (2′-N₃-riboA) and 9-(2′-azido-2′-deoxy-β-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 μM concentration of any studied inosine analog was added to a reaction mixture containing 30 μM inosine (the K_m concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2′-N₃-riboA (K_i=2μM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.     

Inhibition of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase of human erythrocytes by purine and pyrimidine nucleotides.

Orotidine-5′-phosphate decarboxylase of human hemolysates exhibits triphasic kinetics with K_m values of 33, 1.7 and 0.082 μM. Inhibition of this enzyme at low OMP concentrations (<3 μM) by several naturally occurring purine and pyrimidine nucleotides was investigated. No significant inhibition was observed with IMP, GMP, TMP, ADP, and TTP at 5 mM. Inhibition constants for CMP, AMP, and dAMP were 31 μM, 0.11 mM and 0.21 mM, respectively. The results are discussed in relation to inhibition by nucleotides of orotate phosphoribosyltransferase, previously measured with a method which depends on orotidine-5′-phosphate decarboxylase activity.     

8-Amino-9-substituted guanines: potent purine nucleoside phosphorylase (PNP) inhibitors

A series of 8-amino-9-substituted guanines was synthesized and their activity evaluated against human purine nucleoside phosphorylase (PNP). All compounds were found to be potent inhibitors of human PNP (IC50s: 0.17-126 microM). They were also selectively cytotoxic to MOLT-4 lymphoblasts in the presence of a nontoxic amount (10 microM) of the PNP substrate, 2'-deoxyguanosine (GdR). The most potent of these analogs, 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purin-6-one (8-amino-9-(2-thienylmethyl)guanine; PD 119,229) has an IC50 of 0.17 microM (Ki = 0.067 microM), significantly more potent than the known standard, 8-aminoguanosine (IC50 = 1.40 microM). Thus it represents the most potent PNP inhibitor known to date when tested without limiting the concentration of inorganic phosphate.     

Inhibition of liver alcohol dehydrogenase by primaquine and 8-amino-6-methoxyquinoline compounds

The effect of primaquine, chloroquine and other aminoquinoline derivatives on the enzymatic activity of crystalline horse liver alcohol dehydrogenase and partially purified human liver alcohol dehydrogenase has been studied. While chloroquine inhibits neither enzyme, primaquine inhibits both the horse and the human enzyme reversibly and noncompetitively with respect to NAD. The inhibition constant is 2 μM for the horse enzyme and 30 μM for the human enzyme. Spectrophotometric studies of the interaction of primaquine with the horse liver enzyme indicate that primaquine binds to a specific hydrophobic site on the protein. The stoichiometry of binding measured by spectrophotometric titrations is 2 moles primaquine/mole of enzyme and the dissociation constant of the complex is 3 μM. The effects of other 8-amino-6-methoxyquinoline compounds upon horse and human liver alcohol dehydrogenases suggest that the aliphatic side chain, as well as the terminal primary amino group of the side chain, contributes importantly to the tight binding of primaquine to these enzymes.     

Inhibition of dehydrogenase enzymes by hexachlorophene

Low concentrations of the antibacterial agent hexachlorophene (HCP) inhibited a number of pyridine nucleotide-linked dehydrogenase enzymes, including bovine liver glutamate dehydrogenase (GDH), beef heart malate dehydrogenase (MDH), torula yeast glucose 6-phosphate dehydrogenase (G-6-P-D), horse liver alcohol dehydrogenase (ADH), pig heart isocitrate dehydrogenase (ICD), and beef heart lactate dehydrogenase (LDH). Initial velocity studies at appropriate enzyme concentrations gave I₅₀ values for the dehydrogenases which ranged between 1.6 μM for GDH and 105 μM for ICD and LDH. More detailed kinetic analyses of G-6-P-D, ICD and GDH showed that inhibition by HCP in most cases was of the noncompetitive type. Calculations made from the kinetic data gave apparent K_i values with G-6-P-D, of 59 μM for NADP⁺ and of 38 μM for glucose 6-phosphate; with ICD, of 1.0 μM for NADP⁺ and of 25 μM for isocitrate; and, with GDH, of 2.0 μM for NADH, of 7.4 μM for α-ketoglutarate, and of 2.3 μM for ammonium acetate.     

嘌呤核苷酸补偿对吗啡依赖大鼠嘌呤核苷酸分解代谢的影响

目的探讨外源性补偿嘌呤核苷酸对吗啡依赖大鼠嘌呤核苷酸分解代谢的影响。方法将60只♂Wistar大鼠随机分为对照组、吗啡组、吗啡+嘌呤核苷酸组、对照戒断组、吗啡戒断组和吗啡+嘌呤核苷酸戒断组,戒断组于d8给药后4h观察纳洛酮急性戒断症状。各组检测血尿酸(UA)、尿素氮(BUN)和肌酐(Cre)含量,以及血浆和大脑皮质腺苷脱氨酶(ADA)活性。结果吗啡+嘌呤核苷酸戒断组的戒断症状综合评分明显少于吗啡戒断组(P<0.01)。吗啡组及吗啡戒断组血UA含量均明显高于相应的对照组和相应的吗啡+嘌呤核苷酸组(P<0.05)。各组间BUN和Cre含量差异无显著性(P>0.05)。吗啡组及吗啡戒断组血浆及大脑皮质ADA活性明显高于相应的对照组和相应的吗啡+嘌呤核苷酸组(P<0.05或P<0.01)。结论补充嘌呤核苷酸可改善吗啡增强大鼠嘌呤核苷酸分解代谢作用。     

Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides

ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.     

Inhibition of ox brain glutamate dehydrogenase by perphenazine

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the antipsychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However, the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.