Current Medicinal Chemistry (v.20, #34)

The Functional Logic of Cytosolic 5µ-Nucleotidases. by P. L. Ipata, F. Balestri (4205-4216).
Adenosine- and uridine-cytidine kinases, purine-nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase, and several related enzymes, are components of the salvage pathways which reduce the loss of intracellular purine and pyrimidine rings. Although this could explain the role of these enzymes, it poses a problem of the role of the cytosolic 5'-nucleotidase. Why are nucleosides produced from nucleoside-monophosphates, only to be converted back to the same compounds? To date, it is well established that a cross talk exists between the extracellular and intracellular nucleoside metabolism. In districts, such as brain, which are dependent on salvage nucleotide synthesis, nucleosides are produced through the action of the ecto-5'-nucleotidase, the last component of a series of plasma-membrane bound enzyme proteins, catalyzing the successive dephosphorylation of released nucleoside-triphosphates. Both nucleosidetriphosphates (mainly ATP and UTP) and nucleosides (mainly adenosine), act as extracellular signals. Once transported into cell cytosol, all nucleosides are salvaged back to nucleoside-triphosphates, with the exception of inosine, whose salvage is limited to IMP. Intracellular balance of nucleosides is maintained by the action of several enzymes, such as adenosinedeaminase, uridine phosphorylase and cytidine deaminase, and by at least three 5'-nucleotidases, the ADP activated AMP preferring cN-IA, the ATP-ADP activated IMP-GMP preferring cN-II, and the UMP-CMP preferring cN-III. Here we are reviewing the mechanisms whereby cytosolic 5'-nucleotidases control changes in nucleoside and nucleotide concentration, with the aim to provide a common basis for the study of the relationship between biochemistry and other related disciplines, such as physiology and pharmacology.

5'-Nucleotidases, Nucleosides and their Distribution in the Brain: Pathological and Therapeutic Implications by Zsolt Kovács, Árpád Dobolyi, Katalin A. Kékesi, Gábor Juhász (4217-4240).
Elements of the nucleoside system (nucleoside levels, 5'-nucleotidases (5'NTs) and other nucleoside metabolic enzymes, nucleoside transporters and nucleoside receptors) are unevenly distributed in the brain, suggesting that nucleosides have region-specific functions in the human brain. Indeed, adenosine (Ado) and non-Ado nucleosides, such as guanosine (Guo), inosine (Ino) and uridine (Urd), modulate both physiological and pathophysiological processes in the brain, such as sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington's disease, Alzheimer's disease and Parkinson's disease. Interactions have been demonstrated in the nucleoside system between nucleoside levels and the activities of nucleoside metabolic enzymes, nucleoside transporters and Ado receptors in the human brain. Alterations in the nucleoside system may induce pathological changes, resulting in central nervous system (CNS) diseases. Moreover, several CNS diseases such as epilepsy may be treated by modulation of the nucleoside system, which is best achieved by modulating 5'NTs, as 'NTs exhibit numerous functions in the CNS, including intracellular and extracellular formation of nucleosides, termination of nucleoside triphosphate signaling, cell adhesion, synaptogenesis and cell proliferation. Thus, modulation of 5'NT activity may be a promising new therapeutic tool for treating several CNS diseases. The present article describes the regionally different activities of the nucleoside system, demonstrates the associations between these activities and 5'NT activity and discusses the therapeutic implications of these associations.

Nucleoside analogs serve as important chemotherapeutic agents in a number of severe diseases such as cancerand viral infections. These agents are pro-drugs that have to be taken up and phosphorylated in several steps to be trapped in the cells and transformed to active metabolites that inhibit essential steps in the replication of viruses or malignant cells. The anabolic deoxynucleoside kinases (dNKs) and catabolic 5'-nucleotidases(5'-NTs) are involved in maintaining substrate cycles, and act as regulators for the intracellular pools of active nucleotide metabolites. In this chapter the expression patterns of the four dNKs i.e.cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) and the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) as well as the six intracellular 5'-NTs: cN-IA, cN-IB, cN-II, cN-III, cdN, mdN, present in animal cells and tissues will be described. Their role as primary controllers of the accumulation and activation of important anti viral and anti cancer nucleoside analogs in different tissues involved in the pathophysiology of these diseases will be evaluated. The predictability of using the ratios between the activities of the dNKs and 5'-NTs for estimating efficacy and side effects of nucleoside drug candidates will be discussed as well as recommendations on how to use this information to improve future therapies with nucleoside drugs.

A variety of anti-proliferative drugs is based on the structure of purine or pyrimidine nucleosides. These compounds, after phosphorylation, act as analogs of natural nucleotides. In vivo they are recognized by enzymes that transform them either into anti-metabolites targeted to the synthesis of DNA or RNA, or to inactive products of detoxification. 5'-Nucleotidases of different specificity and cellular localization can either remove the phosphate residue from the 5'- position of (deoxy)nucleotide or transfer it from nucleoside monophosphate onto other nucleosides. Such a nucleoside phosphotransferase activity also works with analogs of canonical nucleotides and nucleosides. The majority of nucleoside analogs is metabolized by intracellular cytoplasmic or mitochondrial 5'-nucleotidases and only few reactions proceed on the cell surface. This review summarizes our knowledge of cytoplasmic and mitochondrial forms of 5'-nucleotidases and focuses on their ability to dephosphorylate different analogs of canonical nucleoside 5'-monophosphates. The involvement of 5'-nucleotidases in the phosphotransfer reaction with some nucleoside analogs has been also presented. The importance of the reactions catalyzed by 5'-nucleotidases in clinical resistance to nucleoside-based drugs used in the treatment of cancer or viral diseases, as well as in activation of pro-drugs has been highlighted

Cytosolic 5'-nucleotidase II (cN-II) is an intracellular 5'-nucleotidase characterized by substrate specificity. It preferentially hydrolyzes 6-hydroxypurine nucleotides such as IMP and GMP over AMP or UMP. cN-II is allosterically activated by ATP and inhibited by inorganic phosphate. It also has phosphotransferase activity and transfers phosphate moieties from IMP or GMP to nonphysiological nucleoside analogues used to treat some viral infections or malignancies. The cN-II gene has a strikingly conserved primary structure from humans to nematodes and its activity has been detected in various animals including snails. Its activity is highest in the livers of birds, crocodiles, lizards and snakes. The activity in chicken liver increases 2-fold by feeding a high-protein diet. These results suggest that cN-II participates, through IMP dephosphorylation, in production of uric acid as the main end product of aminonitrogen in these animals. Some studies suggest that cN-II participates in dephosphorylation of IMP accumulated in cells of some tissues to diffusible inosine for reutilization by other tissues. It has also been proposed that cN-II, together with purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase, constitutes the “oxypurine cycle”, thus regulating intracellular phosphoribosyl pyrophosphate (PRPP) concentrations. As for intracellular dephosphorylation of AMP, another intracellular 5'-nucleotidase, cN-I, is supposed to participate, because it hydrolyzes AMP more preferentially than IMP or GMP. However, for the tissues, in which the expression of cN-I is very low or undetectable, e.g. liver or brain tissues, results have been obtained that suggest the participation of cN-II in intracellular dephosphorylation of AMP.

Among the members of the 5'-nucleotidase family, there is only one membrane-bound ectosolic isoenzyme. This esterase prefers AMP as substrate but can hydrolyze a number of purine and pyrimidine phosphorylated compounds, indicating that no evolutive pressure to develop a more restricted specificity was exerted on this enzyme. On the contrary, five cytosolic isoforms have been evolved, probably by convergent evolution, showing different and restricted substrate specificity. The different isoforms have different level of expression and distribution in organs of vertebrates. The cytosolic nucleotidase specific for IMP and GMP (cN-II), is an enzyme allosterically regulated, structurally strongly conserved and expressed at a low but constant level in all organs and tissues in vertebrates. As far as we know, alteration of cN-II expression is limited to pathological conditions. In this review, we report the results of the modulation of cN-II specific activity exerted by silencing or hyperexpression in different cell types, in the attempt to better understand its role and implications in pathology and therapy.

Therapeutic Perspectives for cN-II in Cancer. by Lars Petter Jordheim, Laurent Chaloin (4292-4303).
The cytoplasmic 5'-nucleotidase cN-II is involved in the regulation of endogenous pools of nucleosides andnucleotides together with nucleoside kinases and other intracellular enzymes. A series of results from studies on preclinical models and clinical samples constitutes the basis of the hypothesis in which cN-II is a therapeutic target in cancer. Indeed, the inhibition of its enzymatic activity seems interesting both to induce cell death directly and to increase the anticancer activity of cytotoxic agents used in cancer treatment. Here we will review the current knowledge of the enzymatic function of cN-II together with available structural data and the studies on cN-II in cancer cells and in samples from cancer patients. Recent and ongoing research on cN-II inhibitors is expected to confirm the druggability and the relevance of cN-II as a cancer drug target. Preliminary in vitro data and cancer cell models using cN-II inhibitors have already suggested the pivotal role of this enzyme as therapeutic target allowing the improvement of anticancer treatments.

In mammals, cellular 5'-nucleotidase (5'-NT) activity (EC 3.1.3.5) encompasses a number of genetically and structurally distinct enzyme forms, either membrane-bound or soluble, mainly cytosolic, that are characterized by broad specificity towards nucleoside 5'-monophosphate substrates differing in base (purine/pyrimidine) and/or sugar (oxy/deoxy-ribose) moieties. In particular, among the cytosolic 5'-NTs active towards pyrimidine nucleotides are cN-III and cdN, ubiquitously distributed in mammalian tissues and treated as a single entity in the early days. cN-III was first linked to a genetic defect, hereditary pyrimidine nucleotidase deficiency, associated to a nonspherocytic hemolytic anemia disorder of still unclear mechanism but metabolically characterized by abnormally high levels of pyrimidine compounds and ribonucleoproteins in erythrocytes, as evidenced by occurrence of basophilic stippling on blood smearings. Since the first review on pyrimidine-specific nucleotidases (Amici, A.; Magni, G., Arch. Biochem. Biophys., 2002, 397(2), 184- 190), excellent overviews on the topic appeared in the literature. In the present contribution, the major findings on these two enzymatic proteins, cN-III and cdN, will be described with particular emphasis on the relationships between their structure and function, as well as on their roles in normal and pathological conditions. The catalytic mechanism of both specific hydrolytic and phosphotransferase activities, possessed by both enzymes, will be discussed also in the light of recent solution of both cN-III and cdN three-dimensional structures. This review also focuses on possible therapeutic approaches involving cellular 5'-NTs in detoxifying common antiviral and antineoplastic drugs.