Direct demonstration of the active salvage of preformed purines by murine tumors

The purine de novo biosynthetic pathway has become a target for chemotherapeutic agents and because of the possible contribution of the salvage of extracellular purines to cellular purine pools an examination of the ability of mouse tumors in vivo to exploit the salvage pathways was undertaken. Our data reveal that circulating radiolabeled preformed purines are rapidly and actively salvaged in both normal liver and in two different types of model tumors. The salvaged purines were found to be distributed between both acid soluble cytoplasmic purines and acid insoluble nucleic acid associated purine species. The ability to salvage adenine, the most abundant circulating purine in C57BL/6 mice, was highest in normal liver with the two different model tumors demonstrating lower specific activities of salvaged acid soluble purines. The amount of radiolabel incorporated into acid insoluble nucleic acid was dependent upon the tumor type. Because of the active salvage observed in these tumors, the mechanism by which de novo purine biosynthesis inhibitors serve as effective chemotherapeutic agents may be more complex than simple biosynthetic inhibition.     

Progress toward virtual screening for drug side effects

The development and application of a computational protocol for conducting virtual screens of drug side interactions is described. A conventional drug-docking algorithm (AutoDock) is used to conduct two separate studies. First, a series of docking simulations is performed by using guanosine diphosphate and adenosine diphosphate as prototype drugs with the goal of successfully differentiating known receptors from a large number of bait receptors. Using the electrostatic energy of the purine ring as a basis for discrimination allows the correct identification of receptors in blind studies with 100% specificity and 94% sensitivity. In a second study, similar methodology is used to investigate the binding of clinically relevant inhibitors (Gleevec, purvalanol A, and hymenialdisine) to a variety of protein kinase targets. Overall, excellent agreement between experimental and predicted preferences for kinase targets is obtained. An important conclusion from the latter study is that homology-modeled structures of putative receptors may reasonably be used as targets for docking when directly solved crystal structures are not available. The prospects for routine application of the methodology as a means of identifying potential side interactions of candidate drugs are discussed.     

Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 A resolution

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.

     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

The control of cell proliferation by preformed purines: a genetic study. I. Isolation and preliminary characterization of Chinese hamster lines with single or multiple defects in purine "salvage" pathways.

Sublines with single or multiple defects in purine "salvage" enzymes were isolated from the Chinese hamster fibroblastic line GMA32 through single or successive one-step selections for resistance to purine analogs. They were examined for their ability to incorporate purine bases and nucleosides into macromolecules, for their sensitivity to growth inhibitory purines, and for their rescue by exogenous purines from deprivation imposed by metabolic inhibitors of endogenous synthesis. The results show that a deficiency of either adenosine kinase (EC 2.7.1.20), adenine phosphoribosyltransferase (EC 2.4.2.7) or hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) abolishes the ability of adenine to cause cell death by interfering with pyrimidine synthesis; on the other hand, the pyrimidine starvation caused by adenosine is fully prevented only by a deficiency of adenosine kinase.     

The separation of antagonist from agonist effects of trisubstituted purines on CaV2.2 (N-type) channels

Dihydropyridines can affect L-type calcium channels (CaV1) as either agonists or antagonists. Seliciclib or R-roscovitine, a 2,6,9-trisubstituted purine, is a potent cyclin-dependent kinase inhibitor that induces both agonist and antagonist effects on CaV2 channels (N-, P/Q- and R-type). We studied the effects induced by various trisubstituted purines on CaV2.2 (N-type) channels to learn about chemical structure–function relationships. We found that S-roscovitine and R-roscovitine showed similar potency to inhibit, but agonist activity of S-roscovitine required at least a 20-fold higher concentration, suggesting stereospecificity of the agonist-binding site. The testing of other trisubstituted purines showed a correlation between CaV2.2 inhibition and cyclin-dependent kinase affinity that broke down after determining that a chemically unrelated inhibitor, kenpaullone, was a poor CaV2.2 inhibitor, and a kinase inactive analog (dimethylamino-olomoucine; DMAO) was a strong inhibitor, which together support a kinase independent effect. In fact, like dihydropyridine-induced L-channel inhibition, R-roscovitine left-shifted the closed-state inactivation versus voltage relationship, which suggests that inhibition results from CaV2 channels moving into the inactivated state. Trisubstituted purine antagonists could become clinically important drugs to treat diseases, such as heart failure and neuropathic pain that result from elevated CaV2 channel activity.     

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.     

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate.     

Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7-9 and is characterized by marked stability. Compared with the human enzyme, the recombinant P. falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the Km values for TMP and dGMP are 22 and 30 microM, respectively. The turnover number kcat(TMP) was found to be 3.4 s(-1), a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study, we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.     

Transition state analogue inhibitors of protozoan nucleoside hydrolases

Protozoan parasites are unable to synthesize purines de novo and must rely on purine salvage pathways for their requirements. Nucleoside hydrolases, which are not found in mammals, function as key enzymes in purine salvage in protozoa. Inhibition of these enzymes may disrupt purine supply and specific inhibitors are potential therapeutic agents for the control of protozoan infections. A series of 1,4-dideoxy-1,4-imino-D-ribitols bearing C-bonded aromatic substituents at C-1 have been synthesized, following carbanion additions to the imine 2, and tested as potential nucleoside hydrolase inhibitors. Nucleoside analogues 8, 11, 14, 17, 20, 24-26, 28 exhibit Ki values in the range 0.2-22 microM against two representative isozymes of protozoan nucleoside hydrolases.     

Pyrazolopyrimidine metabolism in Leishmania and trypanosomes: significant differences between host and parasite

The pathogenic hemoflagellates of the genera Leishmania and Trypanosoma are major causes of human disease in the tropical and subtropical areas of the world. In general, the agents used to treat diseases caused by these organisms are toxic and not suitable for administration to the millions of people infected. Investigations over the past several years have shown that there are several major differences between man and these protozoans with respect to purine metabolism. The differences appear to offer promise for the development of effective chemotherapeutic compounds. These organisms do not synthesize purines de novo, as does man. They are able to concentrate pyrazolopyrimidines with the cell and metabolize them as purines through the salvage pathways, ultimately incorporating them into nucleic acids. This does not occur in mammals. The pyrazolopyrimidine base allopurinol, which has served as a prototype, is activated by a phosphoribosyltransferase to the ribonucleotide. The ribonucleotide is aminated to the 4-amino-pyrazolopyrimidine ribonucleotide and subsequently phosphorylated to the triphosphate form and incorporated into RNA. The pyrazolopyrimidine ribonucleosides formycin B and allopurinol ribonucleoside are activated through a nucleoside phosphotransferase. The resulting ribonucleotide is aminated and incorporated into RNA as described above. These metabolic peculiarities occur not only in the forms of these parasites which are found in the insect vectors but also in the intracellular forms which are pathogenic in man. The differences in the enzymology and metabolism of purines which exist in the genera Leishmania and Trypanosoma offer excellent opportunities for chemotherapeutic exploitation.     

Purine Uptake and Utilization by the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte-free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.     

Purines and pyrimidines in malarial parasites

In order for the plasmodium malarial parasite to replicate in the human erythrocyte it requires metabolic pathways which are not operative in the host erythrocyte. Thus, the malarial parasite not only synthesizes enzymes for purine salvage and interconversion, for the pyrimidine biosynthetic pathway de novo, and for the folate cycle, but it also alters the host erythrocyte membrane in respect to the transport of purines. Several of the plasmodium enzymes from these pathways have been cloned and these appear to be highly homologous to the corresponding human enzymes. However, enzymes which have been purified from Plasmodium, have demonstrated physicochemical and kinetic differences and may be potential targets for chemotherapy. Inhibition of individual enzymes, such as the dihydroorotate dehydrogenase (DHO-DHase), and inhibition of the inserted pathway from IMP to AMP and IMP to GMP hold considerable promise as chemotherapeutic targets. An entirely new approach in inhibiting malarial growth involves the altered nucleoside transporter in the infected cell membrane through which cytotoxic compounds may be selectively targeted into only the infected cell.     

Prokaryotes that grow optimally in acid have purine-poor codons in long open reading frames

In nucleic acids the N-glycosyl bonds between purines and their ribose sugar moities are broken under acid conditions. If one strand of a duplex DNA segment were more vulnerable to mutation than the other, then the archaeon Picrophilus torridus, with an optimum growth pH near zero, could have adapted by decreasing the purine content of that strand. Yet, P. torridus has an optimum growth temperature near 60°C, and thermophiles prefer purine-rich codons. We found that, as in other thermophiles, high growth temperature correlates with the use of purine-rich codons. The extra purines are often in third, non-amino acid determining, codon positions. However, as in other acidophiles, as open reading frame lengths increase, there is increased use of purine-poor codons, particularly those without purines in second, amino acid-determining, codon positions. Thus, P. torridus can be seen as adapting (a) to temperature by increasing its purines in all open reading frames without greatly impacting protein amino acid compositions, and (b) to pH by decreasing purines in longer open reading frames, thereby potentially impacting protein amino acid compositions. It is proposed that longer open reading frames, being larger mutational targets, have become less vulnerable to depurination by virtue of pyrimidine for purine substitutions.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.