Donald J. Nelson

Purine metabolism in Leishmania donovani and Leishmania braziliensis.

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Both adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase ((EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors. The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activiting.

Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rete-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine greater than hypoxanthine greater than guanine greater than xanthine.

Formation of nucleotides of [6-14C]allopurinol and [6-14C]oxipurinol in rat tissues and effects on uridine nucleotide pools

Abstract Allopurinol-1-ribonucleotide, oxipurinol-1-ribonucleotide and oxipurinol-7 ribonucleotide have been found in concentrations ranging from less than 10−9-10−6M in rat liver and kidney after administration of [6-14C]allopurinol. The amounts and relative proportions of each nucleotide were dependent upon the dose, route of administration and time. Red cells contained about one-tenth the amount of allopurinol-1-ribonucleotide present in liver and no oxipurinol ribonucleotides were detectable. Brain contained no analog nucleotides. Both of the oxipurinol ribonucleotides were recovered after administration of [6-14Cloxipurinol. In general, the biological half-lives of the nucleotides were related to the amount of the free bases present in the tissues. There was no evidence for the presence of ribonucleoside di- or triphosphates of allopurinol or oxipurinol. Allopurinol and oxipurinol ribonucleotides have been reported to be inhibitors of orotidylate decarboxylase in vitro. High doses of allopurinol and oxipurinol caused a transient decrease of UMP and UDP pools in rat liver, which returned to control levels after 3 hr, whereas UTP levels were actually elevated. In kidney, allopurinol caused no changes in UMP, UDP or UTP levels at any of the times examined, even after a 100 mg/kg, i.p., dose. These findings suggest that control mechanisms can maintain uridine nucleotide levels in a normal range, even in the presence of strong inhibitors of de novo UMP biosynthesis.

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta 544, 360 (1978)]. [8-14C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-14C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form zanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-14C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-14C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).

Separation of 6-thiopurine derivatives on deae-sephadex columns and in the high-pressure liquid chromatograph

Conditions are described for the separation of 6-thiopurine derivatives of pharmacological interest, both on large columns of DEAE-Sephadex A-25 by a modified procedure of Caldwell and in the Varian Aerograph LCS-1000 high-pressure liquid chromatograph. It was found that the triethylammonium acetate buffer, pH 4.7, used by Caldwell caused extensive degradation of 6-thiopurines containing an unsubstituted thiol group, and that this decomposition could be prevented by the addition of 10 mM β-mercaptoethanol. Elution profiles are presented for a number of synthetic 6 thiopurine derivatives investigated by these two chromatographic procedures.

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice.

The pharmacokinetics of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10-100 nmoles [2-3H]adenosine and measurement of blood 3H2O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-3H]adenosine to [2-3H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2-6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.

Effect of acyclovir on the deoxyribonucleoside triphosphate pool levels in Vero cells infected with herpes simplex virus type 1

The effect of acyclovir on the deoxyribonucleoside triphosphate pools of Vero cells infected with herpes simplex virus type 1 was examined. Deoxyguanosine triphosphate and deoxyadenosine triphosphate pool levels in infected cells treated with acyclovir increased dramatically compared with pool levels in untreated infected cels. The increases were due, at least in part, to inhibition of viral DNA polymerase activity which resulted in reduced utilization of the deoxyribonucleoside triphosphates. Differences of as much as 26 times were detected in the sensitivity of herpes simplex virus type 1 to inhibition by acyclovir with different Vero cell cultures. These results were due to differences in acyclovir triphosphate levels, not to differences in deoxyguanosine triphosphate levels.

Ribonucleotides of Allopurinol and Oxipurinol in Rat Tissues and Their Significance in Purine Metabolism

In the early studies on the metabolism of allopurinol in animals and in man [1,2], it was not possible to find any allopurinol or oxipurinol ribonucleotides in acid-soluble extracts of tissues with methods sensitive enough to detect 10 −6M concentrations. Similarly, no such ribonucleotides were detectable in the acid-soluble metabolites of allopurinol in Ehrlich Ascites cells [3] or in human fibroblasts [4]. Although allopurinol was a substrate for hypoxanthineguanine phosphoribosyltransferase (HGPRT) in vitro, the kinetic parameters of allopurinol with human red cell HGPRT [5] indicated that the conditions were not favorable for the formation of this nucleotide in vivo (Table 1). Thus, the Km for allopurinol was 1 mM, whereas a level of 0.01 mM is the highest level attained after therapeutic doses. The binding of oxipurinol to this enzyme was even poorer, Ki = > 10 mM, and the velocity so low that it was not measurable at the 0.125 mM concentration used in the assay [5], nor at 0.5 mM, used subsequently. Moreover, the relatively low Km values of hypoxanthine, and xanthine, and the increased level of these bases resulting from xanthine oxidase inhibition, increased the probability that hypoxanthine and xanthine would successfully compete with their respective analogues for HGPRT under in vivo conditions. Nevertheless, the work of Fox [6] and of Kelley [7,8] indicated that the nucleotides of allopurinol and oxipurinol might indeed be formed and be responsible for the orotic aciduria and orotidinuria seen in allopurinol-treated patients. In order to attempt the quantification of such nucleotides, the present studies in rats were undertaken with large doses of 14C-allopurinol of high specific activity. By the extraction of large amounts of tissue, it was possible to isolate, identify, and quantify the radioactive metabolites. A more detailed account of these investigations will be published elsewhere [9].

Inhibition of xanthine oxidase by 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine

Compound B103U, 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine, was investigated as an inhibitor of human xanthine oxidase. Studies in vitro demonstrated that it was significantly more potent than oxypurinol, 4,6-dihydroxypyrazolo[3,4-d]pyrimidine. It formed an initial complex with electron-rich (reduced) human xanthine oxidase that was tighter than the corresponding complex formed by oxypurinol. The initial complexes with each inhibitor and reduced enzyme were internally rearranged into more stable complexes with first-order rate constants of 2.5 to 3 per min. However, the half-life of the isomerized (stable) complex with B103U was three to four times longer than the half-life of the analogous complex with oxypurinol. This stability was previously noted by Massey et al. (J. Biol Chem 254: 2837-2844, 1970) with B103U and bovine xanthine oxidase. The overall Ki values accounting for the initial and isomerized complexes were 5 nM for B103U and 100 nM for oxypurinol. B103U was also more potent as an inhibitor of bovine xanthine oxidase-catalyzed generation of superoxide radicals. Studies in mice revealed that the relative in vitro potency of B103U was not sustained in vivo. Compared to the inhibition of xanthine oxidase by oxypurinol, inhibition by B103U was neither more potent nor longer lasting. This shortcoming was not caused by weaker inhibition of mouse xanthine oxidase. Instead, it was the result of poor bioavailability. Plasma levels of available B103U rapidly decreased from samples of mouse and human blood because of reversible binding to serum proteins. B103U was also susceptible to oxidation. Two equivalents of H2O2 stoichiometrically oxidized the 6-thiol substituent to a sulfinic acid. This oxidized product was three orders of magnitude weaker as an inhibitor of xanthine oxidase than was B103U.

Oxypurine and 6-Thiopurine Nucleoside Triphosphate Formation in Human Erythrocytes

A variety of oxypurines and 6-thiopurines could be transformed by intact erythrocytes to their nucleoside triphosphate forms when incubations were extended for up to 24 hrs. The specific nucleotide monophosphate kinases which accomplish these reactions in erythrocytes were not identified but their ability to utilize 6-thioIMP, 6-thioXMP and 6-methylthioGMP as substrates, albeit very slowly, is clearly implied by these results. S-methylation of 6-thiopurines was demonstrated in erythrocytes incubated with physiological amounts of methionine-(CH3-3H). 6-Methylthioguanosine triphosphate and 6-methylmercaptopurine riboside triphosphate were formed in micromolar amounts, probably from the corresponding thiopurine nucleotides by methyl transfer from S-adenosylmethionine.