Adenosine kinases

Adenosine kinase catalyzes the transfer of phosphate from ATP to adenosine (Ado) to form AMP and ADP. The separation of the reactants, Ado and ATP, from the products, AMP and ADP, can be accomplished by reversed-phase HPLC (Ci8) with isocratic elution with a mobile phase of 0.1 M potassium phosphate (pH 5.5) and 10% methanol. Detection depends on the substrate. In this assay, it is useful to replace the substrate adenosine with the fluorescent analog formycin A (FoA) and to monitor the column eluent with a fluorescence detector. Thus, ATP and any of its hydrolytic products will not be detected. 

The reaction mixture contained Tris-HCl (pH 7.4) as the buffer, plus ATP, FoA, MgCl, and KC1. The reaction mixture also contained EHNA (erythro-9,2-hydroxy-3-nonyladenine), an inhibitor of the secondary reaction catalyzed by adenosine deaminase. The reaction was started by the addition of the 

The enzyme was prepared from mouse liver. The liver was disrupted by grinding, and any insoluble material was removed by centrifugation at 30,000g for 30 minutes to form an S-30 fraction, which was used throughout the study. 

Of all the nucleoside kinases, the development of adenosine kinase inhibitors has received the most attention. This can be ascribed to the unique role that adenosine plays as a secondary messenger, modulating neuronal activity 

Further analogs of GP515 and GP3269 were explored at Metabasis. Although very potent inhibitors derived from 5-iodotubercidin were reported 

6 nM) and (34) (EC50 = 0.1 nM).66 Gupta and co-workers at McMaster University identified eight non-nucleoside inhibitors of adenosine kinase from a screen of 1040 compounds.67 The most potent of these inhibitors, 8008-6198 

Non-competitive, allosteric inhibitors of adenosine kinase were reported by Butini et al.6S A biological screen of an in-house database of proprietary compounds identified pyrrolobenzoxazepines, representative of a class of nonnucleoside inhibitors of HIV-1 reverse transcriptase, with a unique T shape geometry as micromolar inhibitors of human adenosine kinase (e.g. compound 

A pharmacophore model for new inhibitors was designed by Lee et al., although no inhibitory activity for the proposed inhibitors was determined experimentally.69 Long et al have designed adenosine kinase inhibitors to target Mycobacterium tuberculosis.70,71 Finally, a patent application from McMaster University disclosed phosphonates as millimolar adenosine kinase inhibitors.72 

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Adenosine is formed from ATP via a phosphatase cascade that sequentially involves the diphosphate, ADP, and the monophosphate, AMP. The actions of adenosine are terminated by uptake and rephosphorylation via adenosine kinase to AMP or by cataboHsm via adenosine deaminase to inosine and hypoxanthine. 

Ara-A is phosphorylated in mammalian cells to ara-AMP by adenosine kinase and deoxycytidine kinase. Further phosphorylation to the di- and triphosphates, ara-ADP and ara-ATP, also occurs. In HSV-1 infected cells, ara-A also is converted to ara-ATP. Levels of ara-ATP correlate directly with HSV rephcation. It has recently been suggested that ara-A also may exhibit an antiviral effect against adenovims by inhibiting polyadenylation of viral messenger RNA (mRNA), which may then inhibit the proper transport of the viral mRNA from the cell nucleus. 

Aminoimidazole-4-carboxamide ribonucleoside (also known as AICA riboside or AICAR). An adenosine analogue that is taken up into cells by adenosine transporters and converted by adenosine kinase to the monophosphorylated nucleotide form, ZMP. ZMP is an analogue of AMP that activates the AMP-activated protein kinase (AMPK), for which acadesine or AICAR can be used as a pharmacological activator. 

Unlike classical neurotransmitters, adenosine does not have a rapid synaptic uptake system (as for the biogenic amines), and its chemical inactivation system is not as rapid as for the transmitter acetylcholine, for example. Adenosine may be metabolized extracellularly and inactivated with respect to the ARs in a more general fashion by the widespread enzymes adenosine kinase (AK, to produce AMP) and adenosine deaminase (AD, to produce inosine). Both AMP and inosine are only weakly active at ARs, depending on the subtype. 

The first pharmacological agent shown to activate AMPK was 5-aminoimidazole-4-carboxamide (AICA) riboside, also known as acadesine. This adenosine analogue is taken up into cells by adenosine transporters and phosphoiylated by adenosine kinase to the mono-phosphorylated form, AICA ribotide or ZMP. ZMP accumulates inside cells to higher concentrations than the concentration of AICA riboside present in the medium, and it mimics both effects of AMP on AMPK system (allosteric activation and inhibition of... 

Adenosine kinase catalyzes phosphorylation of adenosine and deoxyadenosine to AMP and dAMP, and de-oxycytidine kinase phosphorylates deoxycytidine and 2 -deoxyguanosine to dCMP and dGMP. 

Fig. 1.13 Adenosine kinase inhibitor designed using fragment optimization approach.

Adenosine metabolism (Fig. 12.2) is reviewed in Dunwiddie Masino (2001) and Ribeiro et al. (2002). The phosphorylation of intracellular adenosine to AMP is catalyzed by adenosine kinase. Intracellularly, adenosine can also be deami-nated to inosine by adenosine deaminase. Free intracellular adenosine is normally low. Excess adenosine, which cannot be regenerated to ATP, is extruded to the extracellular space by equilibrative nucleoside transporters (ENTs) in the cell membrane. During electrical stimulation or energy depletion, adenosine is... 

Nitric oxide (NO) is an intercellular signaling molecule that can inhibit neuronal energy production (Brorson et al., 1999 Malefic et al., 2004). It has been found that NO donors cause large increases in extracellular adenosine in cultures of forebrain neurons (Rosenberg et al., 2000). These were shown to be caused by NO release, and the accumulation of adenosine was not blocked by probenecid (ENT blocker) or GMP (a blocker of AMP hydrolysis), suggesting that adenosine was likely of intracellular origin. Indeed, it was found that NO donors caused a decrease in intracellular ATP and the inhibition of adenosine kinase activity, possibly due to the rise in adenosine. 

As mentioned above, inhibition of adenosine kinase increases extracellular adenosine concentrations. Interest in enhancement of the neuroprotective, antinociceptive, and anti-inflammatory actions of adenosine has encouraged development of systemically applicable adenosine kinase inhibitor drugs. For example, it was found recently that the specific adenosine kinase inhibitor ABT-702, when given intraperitoneally, increases sleep and slow wave EEG activity of rats (Radek et al, 2004), a finding that encourages further research into the hypnogenic effect of this type of drug. 

Alanko, L., Heiskanen, S., Stenberg, D. Porkka-Heiskanen, T. (2003a). Adenosine kinase and 5 -nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat. Neurochem. Int. 42 (6), 449-54. 

Lloyd, H. G. Fredholm, B. B. (1995). Involvement of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration in rat hippocampal slices. Neurochem. Int. 26 (4), 387-95. 

Pak, M. A., Haas, H. L., Decking, U. K. Schrader, J. (1994). Inhibition of adenosine kinase increases endogenous adenosine and depresses neuronal activity in hippocampal slices. Neuropharmacology 33 (9), 1049-53. 

Rosenberg, P. A., Li, Y., Le, M. Zhang, Y. (2000). Nitric oxide-stimulated increase in extracellular adenosine accumulation in rat forebrain neurons in culture is associated with ATP hydrolysis and inhibition of adenosine kinase activity. 

Sciotti, V. M. Van Wylen, D. G. (1993). Increases in interstitial adenosine and cerebral blood flow with inhibition of adenosine kinase and adenosine deaminase. J. Cereb. Blood Flow Metab. 13 (2), 201-7. 

Yamada, Y., Goto, H. 8r Ogasawara, N. (1980). Purification and properties of adenosine kinase from rat brain. Biochim. Biophys. Acta 616 (2), 199-207. 

The biosynthesis of adenosine is theoretically controlled by several processes namely (1) the biosynthesis of adenosine from AMP by 5 -nucleotidase [EC 3.1.3.5], (2) from S-adenosyl homocysteine by S-adenosyl homocystine hydrolase [EC 3.3.1.1], (3) the metabolism of adenosine to AMP by adenosine kinase [EC 2.7.1.20], and (4) to inosine by adenosine deaminase (ADA) [EC 3.5.4.2], Interestingly, both 5 -nucleotidase and ADA activities were found to be highest in the leptomeninges of rat brain in contrast, the adenosine kinase activity was widely distributed throughout the brain parenchyma, which has negligible ADA activity... 

Sinciair, C. J., et al. Nucleoside transporter subtype expression effects on potency of adenosine kinase inhibitors. Br. J. Pharmacol. 2001, 134, 1037-1044. 

T. brucei is unable to synthesize purines de novo and, as such, is dependent upon salvage mechanisms from the host. A number of transporters and enzymes are used by T. brucei to accomplish this task, and inhibition of these targets offers promise for development of trypanocides [39]. This strategy has been validated by demonstration that cordycepin (34), a substrate for T. brucei adenosine kinase (TbAK), which terminates RNA synthesis and parasite growth, can cure stage 2 HAT infections in mice when coadministered with deoxycoformycin (35), an adenosine deaminase inhibitor [40]. 

ATP diphosphohydrolase Diadenosine polyphosphatase 5 nucleotidase Nucleoside transporter Adenosine deaminase Adenosine kinase Xanthine oxidase Nucleoside phosphorylase... 

Adenosine is not a classical neurotransmitter because it is not stored in neuronal synaptic granules or released in quanta. It is generally thought of as a neuromodulator that gains access to the extracellular space in part from the breakdown of extracellular adenine nucleotides and in part by translocation from the cytoplasm of cells by nucleoside transport proteins, particularly in stressed or ischemic tissues (Fig. 17-2C). Extracellular adenosine is rapidly removed in part by reuptake into cells and conversion to AMP by adenosine kinase and in part by degradation to inosine by adenosine deaminases. Adenosine deaminase is mainly cytosolic but it also occurs as a cell surface ectoenzyme. 

Adenosine triphosphate (ATP) is one of the most important cofactors involved in many of the synthetic reactions going on within the cell. Its recent large scale in vitro enzymatic synthesis from adenosine and acetylphosphate is of particular interest. Three enzymes immobilized in polyacrylamide gel were used adenosine kinase, adenylate kinase and acetate kinase (lip. ... 

The purine and pyrimidine bases can be converted to then-respective nncleotides by reaction with 5-phosphoribosyl 1-pyrophosphate. Since these bases are not very soluble, they are not transported in the blood, so that the reactions are only of qnantitative significance in the intestine, where the bases are produced by degradation of nucleotides. In contrast, in some cells, nucleosides are converted back to nucleotides by the activity of kinase enzymes. In particular, adenosine is converted to AMP, by the action of adenosine kinase, and uridine is converted to UMP by a uridine kinase... 

Severe combined immunodeficiency disease The enzyme adenosine deaminase degrades deoxyadenosine which is produced during DNA degradation (Chapter 10). Deficiency of the enzyme results in accumulation of deoxyadenosine which is a substrate for adenosine kinase and leads to production of deoxyadenosine and deoxyquanosine triphosphates, which reach high concentrations. This disturbs the balance of deoxy nucleotides which results in failure of DNA replication. This enzyme is normally present in lymphocytes so that a deficiency prevents proliferation of the lymphocytes, which is essential in combatting an infection. Consequently, patients are very susceptible to infections. This is one disease that is effectively treated by gene therapy. 

Table 2.2. SUBSTRATE SPECIFICITY OF ADENOSINE KINASE A. Variation of substituents on purine ring... 

Fluoroadenine, 2-chloroadenine, 2-aminoadenine, 2- and 8-aza-adenines, 4-aminopyra7.olo[3, 4-d] pyrimidine and 6-methylpurine are converted to their ribonucleosidesby adenosine phosphoribosyltransferase,and their ribonucleosides are converted to the ribonucleotides by adenosine kinase most of the ribonucleotides are then converted to the di- and triphosphates. A -Aminoadenine, A -hydroxyadenine, A -methyladenine, purine, 7-deaza-adenine, and 7-amino-pyrazolo[4, 3-d] pyrimidine are either not substrates or are very poor substrates for the phosphoribosyltransferase, but their ribonucleosides are excellent substrates for the kinase. The ribonucleotides of purine, 7-deaza-adenine and... 

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