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Query: UNIPROT:P06889 (Mol)
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Full and partial agonists of the A1 adenosine receptor were characterized with respect to their influence on G protein activation and their thermodynamic parameters of receptor binding in rat brain membranes. G protein activation was determined through measurement of [35S]guanosine-5'-(gamma-thio)triphosphate ([35S]GTP[S]) binding, and receptor binding was studied under identical conditions through the displacement of [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) in equilibrium binding studies. The intrinsic activity in stimulating [35S]GTP[S] binding did not correlate with the affinity of the ligands. 5'-Deoxy-5'-methylthioadenosine, 2-phenylaminoadenosine, and 2-chloro-2'-deoxyadenosine were identified as partial A1 agonists in the G protein activation assay. Depending on the temperature, these ligands showed agonistic and antagonistic properties to varying extents. EC50 values for G protein stimulation and KH and KL values of the partial agonists decreased when the incubations were performed at lower temperatures, indicating a mainly enthalpy-driven process of interaction with the receptor. Thermodynamic parameters of receptor binding of the partial agonists resembled the characteristics of the antagonist DPCPX more closely than those of the agonist 2-chloro-N6-cyclopentyladenosine. In addition, partial agonists detected fewer A1 adenosine receptors in the high affinity state than did full agonists. The lower efficacy in stimulation of the binding of [35S]GTP[S] is probably the consequence of an impaired ability of the partial agonists to release GDP from the G protein, as was shown by an impaired release of prebound [35S]GDP[S] from the membranes.
Mol Pharmacol 1996 May
PMID:Interaction of full and partial agonists of the A1 adenosine receptor with receptor/G protein complexes in rat brain membranes. 862 42

The efficacy of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against the replication of human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1) and its cellular metabolism were investigated in human primary macrophages from seronegative donors. PMEA potently inhibited the replication of both HIV and HSV-1 in macrophages, with similar EC50 values (0.025 and 0.032 microM, respectively), whereas the EC50 values of PMEA in lymphocytic C8166 cells and fibroblastoid Vero cells were 150-200-fold higher (3.5 and 7.9 microM, respectively). Granulocyte/macrophage colony-stimulating factor and macrophage colony-stimulating factor, two cytokine enhancers of the replication of HIV (and HSV-1), decreased the activity of PMEA against both viruses, yet EC50 values were still lower than in lymphocytes and fibroblasts. Thus, the selectivity index of PMEA in macrophages was > 2 orders of magnitude higher than that in lymphocytes and fibroblasts and still > 1 log higher under conditions of enhancement of virus replication in macrophages. The intracellular levels of 2'-deoxyadenosine-5'-triphosphate, the natural competitor of PMEA-diphosphate at the level of viral DNA polymerase (either RNA or DNA dependent), were 5-12-fold lower in macrophages than in other cells. Furthermore, intracellular concentrations of PMEA-diphosphate (the active metabolite of PMEA) were unusually much higher in macrophages (with or without cytokines) than in lymphocytes and fibroblasts. Consequently, the ratio of PMEA-diphosphate to 2'-deoxyadenosine-5'-triphosphate in monocytes/macrophages was approximately 2 orders of magnitude higher in macrophages than in the other cells and correlated closely with the pronounced antiviral potency of PMEA. The dual potent activity of PMEA against HIV and HSV-1 stresses the importance of clinical trials to assess the role of this drug in the therapy of HIV-related disease.
Mol Pharmacol 1996 Aug
PMID:Potent inhibition of human immunodeficiency virus and herpes simplex virus type 1 by 9-(2-phosphonylmethoxyethyl)adenine in primary macrophages is determined by drug metabolism, nucleotide pools, and cytokines. 870 Jan 44

2-Chloro-2'-deoxyadenosine [CldAdo (cladribine)], a novel effective antileukemic agent, was examined for its effects on cellular mitochondrial function and DNA content after long term (< or = 7-day) incubation of cultured CCRF-CEM human leukemia cells. Dideoxycytidine (ddC), which is known to have a delayed effect on mitochondrial DNA content, was used as a positive control to monitor mitochondrial dysfunction. CldAdo at 6-16 nM was toxic to cells within 24 hr, which is in contrast to 300 nM ddC, which had no effect on cell growth for the first 4 days of treatment. Cellular lactic acid production was used to monitor concomitant perturbations in oxidative phosphorylation during drug treatment. Unlike the delayed increase in lactate observed with ddC exposure, CldAdo-treated cells exhibited a 2-2.4-fold increase in lactate levels after 2 days of exposure to 16 nM CldAdo. By days 4 and 7, however, lactate production returned to control levels. Shorter incubations with CldAdo revealed that lactate levels began to increase within 12 hr of drug exposure, paralleling cytotoxicity. We also examined mitochondrial DNA content during drug treatment by competitive polymerase chain reaction. ddC (300 nM) reduced mitochondrial DNA levels from approximately 1000 copies/untreated cell to approximately 130 copies/cell after 7 days of exposure. In contrast, cytotoxic doses of CldAdo had little or no effect on mitochondrial DNA content during the 1-week incubation. Thus, the early CldAdo-induced perturbation of mitochondrial function was not associated with a loss of mitochondrial DNA per cell. In addition, no evidence of DNA laddering, indicative of cellular apoptosis, was detected at these dosage levels and treatment times.
Mol Pharmacol 1997 Apr
PMID:2-Chloro-2'-deoxyadenosine, an antileukemic drug, has an early effect on cellular mitochondrial function. 910 26

Adenosine 3',5'- and 2',5'-bisphosphates previously were demonstrated to act as competitive antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329). 2'- and 3'-Deoxyadenosine bisphosphate analogues containing various structural modifications at the 2- and 6-positions of the adenine ring, on the ribose moiety, and on the phosphate groups have been synthesized with the goal of developing more potent and selective P2Y1 antagonists. Single-step phosphorylation reactions of adenosine nucleoside precursors were carried out. The activity of each analogue at P2Y1 receptors was determined by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit phospholipase C stimulation elicited by 10 nM 2-MeSATP (antagonist effect). Both 2'- and 3'-deoxy modifications were well tolerated. The N6-methyl modification both enhanced antagonistic potency (IC50 330 nM) of 2'-deoxyadenosine 3',5'-bisphosphate by 17-fold and eliminated residual agonist properties observed with the lead compounds. The N6-ethyl modification provided intermediate potency as an antagonist, while the N6-propyl group completely abolished both agonist and antagonist properties. 2-Methylthio and 2-chloro analogues were partial agonists of intermediate potency. A 2'-methoxy group provided intermediate potency as an antagonist while enhancing agonist activity. An N1-methyl analogue was a weak antagonist with no agonist activity. An 8-bromo substitution and replacement of the N6-amino group with methylthio, chloro, or hydroxy groups greatly reduced the ability to interact with P2Y1 receptors. Benzoylation or dimethylation of the N6-amino group also abolished or greatly diminished the antagonist activity. In summary, our results further define the structure-activity of adenosine bisphosphates as P2Y1 receptor antagonists and have led to the identification of the most potent antagonist reported to date for this receptor.
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PMID:Deoxyadenosine bisphosphate derivatives as potent antagonists at P2Y1 receptors. 945 42

Deoxyguanosine kinase (dGK) is an enzyme responsible for the phosphorylation of purine deoxynucleosides in mitochondria of mammalian cells. Its role in activation of pharmacologically used nucleoside analogs is not well understood, because of the low levels of dGK found in tissue extracts and its inactivation during purification. The cDNA for dGK was recently cloned and expressed in Escherichia coli. Here we present an improved procedure for expression and purification of a highly active form of human recombinant dGK. The enzyme showed a broad substrate specificity toward natural purine and pyrimidine deoxynucleosides as well as toward important nucleoside analogs. The Km and Vmax values for deoxyguanosine, deoxyinosine, deoxyadenosine, and deoxycytidine were 4, 13, 460, 330 microM and 43, 330, 430 and 60 nmol/min/mg of protein, respectively. Antileukemic purine analogs such as arabinosyl guanine, 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, and 2-fluoro-arabinosyl-adenine were phosphorylated as efficiently by dGK as the natural nucleoside substrates. This is the first report in which 2-fluoro-arabinosyl-adenine and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine were shown to be good substrates for dGK. The antiviral analogs dideoxyinosine and arabinosyl adenine also showed significant activity with dGK, as did several pyrimidine analogs (e.g., the cytostatic drugs 5-fluoro-2'-deoxycytidine and difluorodeoxycytidine). The broad specificity of dGK described here may change our understanding of the mechanisms responsible for the efficacy and mitochondrial toxicity of several nucleoside analogs.
Mol Pharmacol 1998 Feb
PMID:Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimidine analogs. 946 85

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked 3H-adenosine uptake by oocyte-cumulus cell complexes (OCCs) and cumulus cell-enclosed oocytes (CEOs) by 82-85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis-arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine-metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5'-amino-5'-deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine +/- adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis-inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis-arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine-mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine.
Mol Reprod Dev 1999 Jun
PMID:Uptake and metabolism of adenosine mediate a meiosis-arresting action on mouse oocytes. 1033 59

Adenosine influx by Trypanosoma brucei brucei P1 and P2 transporters was kinetically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K(m) = 0.38 +/- 0.10 microM, V(max) = 2.8 +/- 0.4 pmol x 10(7) cells(-1) x s(-1)) than the P2 transporter (K(m) = 0.92 +/- 0.06 microM, V(max) = 1.12 +/- 0.08 4 pmol x 10(7) cells(-1) x s(-1)). To formulate a structure-activity relationship for the interaction of adenosine with the transporters, a series of analogs were evaluated as potential inhibitors of adenosine transport, and the K(i) values were converted to binding energy. The P1 transporter was found to be selective inhibited by purine nucleosides (K(i) approximately 1 microM for inosine and guanosine), but nucleobases and pyrimidines had little effect on P1-mediated transport. The P1 transporter appears to form hydrogen bonds with N3 and N7 of the purine ring as well as with the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond energies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-affinity (K(i) = 0.2-4 microM) for 6-aminopurines, including adenine, 2'-deoxyadenosine, and tubercidin, but not for any oxopurines. The main interaction of adenosine with the P2 transporter is suggested to be via hydrogen bonds to N1 and the 6-amino group. Additional pi-pi interactions of the purine ring and electrostatic interactions with N9 may also be important. The predicted substrate recognition motif of P2, but not of P1, corresponds to parts of the melaminophenylarsenical and diamidine molecules, confirming the potent inhibition observed with these trypanocides for P2-mediated adenosine transport (K(i) = 0.4-2.4 microM).
Mol Pharmacol 1999 Dec
PMID:Adenosine transporters in bloodstream forms of Trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs. 1057 43

Oxyamines such as hydroxylamine and methoxylamine disturb DNA replication and act as potent mutagens, causing nucleotide transition from one purine to another or one pyrimidine to another. In order to investigate mismatch base-pairing in DNA damaged with oxyamines, a dodecamer with the sequence d(CGCGmo(6)AATCCGCG), where mo(6) A is 2'-deoxy-N(6)-methoxyadenosine, was synthesized and its crystal structure determined. No significant conformational changes are found between the present dodecamer and the original undamaged B-form dodecamer. Electron density maps clearly show that the mo(6)A residue forms a base-pair with a 2'-deoxycytidine residue through hydrogen bonds similar to a Watson-Crick G.C base-pair. For these hydrogen bonds to be made, N(6)-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result, misincorporation of 2'-deoxycytidine instead of thymidine, or 2'-deoxyadenosine instead of 2'-deoxyguanosine, can occur in DNA replication.
J Mol Biol 1999 Dec 17
PMID:Crystallographic studies on damaged DNAs. I. An N(6)-methoxyadenine residue forms a Watson-Crick pair with a cytosine residue in a B-DNA duplex. 1060 Mar 79

Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2'-deoxyadenosine were 23 and 16 microM, respectively. Purine riboside was a competitive inhibitor with Ki of 5 microM, whereas 2'-3'-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 microM). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg2+ (100 microM) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, alpha-adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3'-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2'-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.
Mol Cell Biochem 2000 Jan
PMID:Purification and characterization of intestinal adenosine deaminase from mice. 1071 33

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.
J Mol Med (Berl) 2001 Apr
PMID:Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast. 1135 35


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