Antitrypanosomal effect of allopurinol: conversion in vivo to aminopyrazolopyrimidine nucleotides by Trypanosoma curzi.

The parasite Trypanosoma cruzi metabolizes allopurinol by a sequential conversion to allopurinol mononucleotide and aminopurinol mononucleotide. The latter is incorporated into RNA. This transformation of a widely used innocuous agent, allopurinol, into a toxic adenine analog appears to account for the antiprotozoan effect of allopurinol. These unique enzymatic activities appear to occur only in T. cruzi and the pathogenic lesihaminae. Allopurinol may serve as a model for the synthesis of similar antiprotozoan agents.

Purine metabolism in the bloodstream forms of Trypanosoma gambiense and Trypanosoma rhodesiense.

Bloodstream forms of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine greater than inosine greater than guanosine greater than xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine greater than adenine greater than guanine greater than xanthine.

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.

Pyrazolopyrimidine metabolism in the pathogenic trypanosomatidae.

Pyrazolopyrimidines are purine analogues. These compounds are metabolized by the pathogenic hemoflagellates and other members of the family Trypanosomatidae as though they were purines. This metabolic sequence does not exist in man or other mammals. In the hemoflagellates, the pyrazolopyrimidine base, of which allopurinol is the paradigm, undergoes ribosylphosphorylation to the ribonucleotide. This ribonucleotide may remain as such or be aminated to the amino analogue and further converted to the aminopyrazolopyrimidine ribonucleoside triphosphate. The latter is incorporated into RNA. This metabolic sequence has been demonstrated in the genera Leishmania and Trypanosoma.

Pyrazolopyrimidine metabolism in African trypanosomes: metabolic similarities to Trypanosoma cruzi and Leishmania spp.

Growth inhibition and radioisotope incorporation studies with allopurinol (4-hydroxypyrazolo(3,4-d)pyrimidine) have shown that the African trypanosomes are biologically and biochemically similar to Leishmania spp. and Trypanosoma cruzi with respect to their response to this compound. These organisms, as a group, share the unique ability to convert allopurinol sequentially to its ribonucleoside monophosphate and 4-aminopyrazolo(3,4-d)pyrimidine ribonucleoside mono-, di- and triphosphates.

Purine metabolism in Leishmania donovani amastigotes and promastigotes.

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.

The activity of ketoconazole and other azoles against Trypanosoma cruzi: biochemistry and chemotherapeutic action in vitro.

Trypanosoma cruzi epimastigotes in culture medium, and amastigotes and trypomastigotes in cultured human diploid lung cells were exposed to the antimycotic agent ketoconazole and their growth and/or sterol biosynthesis observed. Propagation of epimastigotes and amastigotes was impaired by concentrations of ketoconazole achievable in human serum, and amastigotes were more sensitive than were epimastigotes. Epimastigotes and trypomastigotes (non-dividing stage) displayed changes in their membrane sterol content such that the amounts of normal, end-product sterols (ergosterol, ergosta-5,7-dien-3 beta-ol, 24-ethylcholesta-5,7,22-trien-3 beta-ol, 24-ethylcholesta-5,7-dien-3 beta-ol) were notably decreased and the amounts of 14 alpha-methyl sterol precursors of these sterols (24-methylenedihydrolanosterol, obtusifoliol, lanosterol) were increased. Other azole drugs, itraconazole and fluconazole, when tested on epimastigotes, evoked the same qualitative pattern of changes in free sterols. Itraconazole was nearly as potent as ketoconazole, but fluconazole was significantly less potent. The nature of the sterols found in T. cruzi and the actions of azole drugs on their biosynthesis were similar in many respects to those observed in fungi and in Leishmania species. By analogy, it would seem that the primary mechanism of action of azole drugs on T. cruzi life-cycle stages is the impairment of the cytochrome P-450 sterol 14 alpha-demethylase. The consequent loss of normal sterols and accumulation of 14 alpha-methyl sterols may be responsible for the coincident retardation or cessation of growth.

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.

Synthesis, structure, and antiparasitic activity of sulfamoyl derivatives of ribavirin.

The triazole nucleoside derivatives 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-carboxamide (2), 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-thiocarboxamide (3), and 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl)-[1,2,4]triazole-3- carbonitrile (4) were synthesized. Suitably protected triazole nucleosides were converted to their corresponding 5'-sulfamoyl derivatives, which on subsequent deprotection gave the desired compounds in good yields. The structures of compounds 2-4 were confirmed by X-ray crystallographic analysis. All three compounds showed significant antiparasitic activity in vitro, while 2 showed significant activity in vivo against Leishmania donovani and Trypanosoma brucei.

Adenosine analog metabolism in Giardia lamblia. Implications for chemotherapy.

Certain adenosine analogs can inhibit the growth of Giardia lamblia. This biological action correlates with the ability of the organism to phosphorylate the nucleoside directly to the nucleotide. Four of these, 8-azaadenosine, 1-deazaadenosine, 7-deazaadenosine, and 9-deazaadenosine, were very effective. The respective bases of the first three were ineffective. The base of 9-deazaadenosine was not tested as this C-nucleoside is non-cleavable. Metabolic studies using radioactive 7- and 9-deazaadenosine showed that these compounds were phosphorylated by the organism. Enzymatic assay confirmed the presence of nucleoside phosphotransferase activity; no nucleoside kinase activity was found. Preliminary characterization of this phosphotransferase suggests that it has different substrate and phosphate donor specificities than the mammalian enzyme and, therefore, may be a potential site for chemotherapeutic attack.

Adenylosuccinate synthetase and adenylosuccinate lyase from Trypanosoma cruzi, Specificity studies with potential chemotherapeutic agents.

Adenylosuccinate (succino-AMP) synthetase and succino-AMP lyase isolated from epimastigotes of Trypanosoma cruzi by chromatography on phosphocellulose. The synthetase was capable of catalyzing the condensation of aspartic acid with IMP and several IMP analogs. The reaction with allopurinol ribonucleotide is of potential chemotherapeutic interest. This analog was slowly converted to its corresponding succino-AMP analog with a Km' of 140 micrometers (cf. IMP at 10 micrometers) and a Vmax' of 0.3 per cent the rate with IMP. The comparable reaction with this analog does not occur with succino-AMP synthetase from a representative mammalian source [T. Spector and R. L. Miller, Biochim. biophys. Acta 455, 509 (1976)]. The protozoal succino-AMP lyase had a broad substrate which was characteristic of this enzyme from many sources. It catalyzed the rapid and efficient cleavage of all the succino-AMP analogs that were produced by succino-AMP synthetase. Thus, these two enzymes appear to be responsible for the selective amination of allopurinol ribonucleotide in T. cruzi. The metabolically produced AMP analog may be the agent or a precursor of the agent that accounts for the anti-growth activity of allopurinol in these organisms. Similar selective amination was observed previously with these enzymes from Leishmania donovani [T. Spector, T. E. Jones and G. B. Elion, J. biol. Chem. 254, 8422 (1979)]. Thiopurinol ribonucleotide was not a substrate of succino-AMP synthetase from T. cruzi, but it was an inhibitor with a K1 = 33 micrometers. Therefore, the weakness of thiopurinol's anti-growth activity with T. cruzi is not due to its inability to inhibit this enzyme.

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).

Antiprotozoal activity of 3'-deoxyinosine. Inverse correlation to cleavage of the glycosidic bond.

Two nucleosides related to the known antiprotozoal agent 1-(beta-D-ribofuranosyl)-1,5-dihydro-4H-pyrazolo-[3,4-d]pyrimidine-4-one (allopurinol riboside, 1) were prepared and evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. 3'-Deoxyinosine (2) exhibited potent antiprotozoal activity against the three protozoal pathogens with minimal toxicity for host cells. It was found to be especially effective against the Columbia strain of T. cruzi reported to be resistant to 1. The antiprotozoal activity of 2 appeared to be inversely related to the rate of cleavage of the glycosidic bond, as shown by metabolic profiles of 2 in the various pathogenic hemoflagellates and host cells. Combining the key structural elements of 1 and 2 led to the synthesis of 1-(3-deoxy-beta-D-erythro-pentofuranosyl)-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidin-4-one (3'-deoxy-allopurinol riboside, 3). which was found to be inactive as an antiprotozoal agent.

Antileishmanial action of 4-thiopyrazolo (3.4-d) pyrimidine and its ribonucleoside. Biological effects and metabolism.

Thiopurinol [4-thiopyrazolo(3.4-dyprimidine, TPP] and its ribonucleoside (TPPR) were effective in vitro against the intracellular and extracellular forms of L. braziliensis and L. mexicana. They also inhibited the transformation of the amastigote of L. donovani to the promastigote. These thio-analogues had about the same activity as allopurinol [4-hydroxypyrazolo(3.4-d)pyrimidine, HPP] and its ribonucleoside (HPPR). the thiopyrazolopyrimidines were converted primarily to the ribonucleoside-5' -phosphate (TPPR-MP) and to an unidentified metabolite, but not to any of the adenine ribonucleoside analogues previously shown to be formed from allopurinol and its ribonucleoside. There was an antagonism between the growth-inhibitory effects of allopurinol and thiopurinol. This is consistent with the findings that the intracellular concentrations of TPP and TPPR-MP are sufficient to inhibit the conversion of allopurinol to allopurinol ribonucleotide (HPPR-MP) by the hypoxanthine-guanine phosphoribosyltransferase by 30 per cent and the amination of HPPR-MP by adenylosuccinate synthetase by 50 per cent respectively. Consequently, the incorporation of the aminated product (aminopyrazolopyrimidine) into RNA was substantially decreased. The difference in metabolism between the thio- and hydroxypyrazolopyrimidines suggests a difference in their mechanisms of action against the pathogenic leishmania.

Monophosphates of formycin B and allopurinol riboside. Interactions with leishmanial and mammalian succino-AMP synthetase and GMP reductase.

Formycin B 5'-monophosphate (Form B-MP) and allopurinol riboside 5'-monophosphate ( HPPR -MP) are isomers of IMP that are metabolically produced when Leishmania spp. are incubated with the antileishmanial agents formycin B and allopurinol or allopurinol riboside. The interactions of Form B-MP with succino -AMP synthetase and GMP reductase from both leishmanial and mammalian sources were compared with the data of earlier studies with HPPR -MP. Both analogs could substitute for IMP as a substrate for succino -AMP synthetase isolated from Leishmania donovani. The V'max values of Form B-MP and HPPR -MP were about 1% of the V'max of IMP. Only Form B-MP (and not HPPR -MP) could serve as an alternative substrate for mammalian succino -AMP synthetase. The V'max of Form B-MP was 40% that of IMP. The corresponding analogs of AMP, ADP and ATP were produced when Formycin B was incubated with mouse L cells. The Formycin A residue was incorporated into the cellular RNA. The amount of Formycin A-TP produced (relative to ATP) in mouse L cells was considerably less than that produced in Leishmania spp. Both Form B-MP and HPPR -MP were inhibitors of partially purified GMP reductase from L. donovani. The binding of Form B-MP and HPPR -MP to human GMP reductase was 40- and 100-fold weaker, respectively, than the binding to leishmanial GMP reductase. Pretreatment of promastigotes of L. donovani with either allopurinol or Formycin B resulted in greater than 95% reduction of the incorporation of the radiolabel from [14C]xanthine into ATP and greater than 80% reduction of the incorporation of the label into GTP. The HPPR -MP and Form B-MP present in these cells may have inhibited the leishmanial succino -AMP synthetase and GMP reductase. The analogs had little or no effect on the pool sizes of ATP and GTP of either mouse L cells or L. donovani.

Endotoxin disrupts beta-adrenergic signal transduction in the heart.

BACKGROUND: Nonsurvivors of septic shock demonstrate impaired myocardial function refractory to the administration of beta-agonists. METHODS: Using the isolated rat heart preparation, the integrity of the beta-adrenergic transduction pathway was tested (rate pressure product, rate of contraction, rate of relaxation, and cyclic adenosine monophosphate content) using isoproterenol hydrochloride (beta-receptor agonist) or colforsin (forskolin) (adenylyl cyclase activator) stimulation following intracoronary endotoxin infusion. RESULTS: Basal rate pressure product, rate of contraction, rate of relaxation, and cyclic adenosine monophosphate concentrations were unaffected by endotoxin infusion. Endotoxin impaired, increases in rate pressure product, rates of contraction and relaxation, and cyclic adenosine monophosphate to isoproterenol (P < .05), but the response to colforsin was unaffected by endotoxin. CONCLUSIONS: Endotoxin disrupts the myocardial response to direct beta-receptor stimulation but not to adenylyl cyclase stimulation in the isolated rat heart. CLINICAL RELEVANCE: Alteration of the proximal beta-adrenoreceptor complex by endotoxin suggests that therapy of the failing heart during refractory septic shock may be directed to intact sites distal in the beta-adrenergic pathway.

The synergistic action of pyrazolopyrimidines and pentavalent antimony against Leishmania donovani and L. braziliensis.

Pyrazolopyrimidines, particularly allopurinol, allopurinol riboside, and other purine analogues, show promise as experimental therapeutic compounds for the treatment of leishmaniasis. The combination of these agents with pentostam may produce an improved therapeutic effect. We report here on strong synergistic activity between pyrazolopyrimidines and pentavalent antimonials in a human macrophage tissue culture system infected with Leishmania donovani and L. braziliensis.

Prevention of transfusion-induced Chagas' disease by amphotericin B.

Amphotericin B, a polyene antibiotic effective against eukaryotic cells, can eliminate the trypomastigote form of Trypanosoma cruzi from blood stored at 4 degrees C. This antitrypanosomal effect can be achieved with a concentration of 3 micrograms/ml within 48 hours. This concentration of amphotericin B does not produce hemolysis over a period of 3 weeks. Amphotericin B methyl ester and nystatin are not effective. Amphotericin B may be considered as a replacement for crystal violet in blood bank blood to prevent transfusion-induced Chagas' disease.

Regulation of aerobic fermentation in protozoans. VI Comparative biochemistry of pathogenic and nonpathogenic protozoans.

We have investigated the oxidation of carbohydrate by several protozoan organisms. The oxidation is incomplete and results in a mixture of organic acid and carbon dioxide. The phosphofructokinase and pyruvate kinase, enzymes normally under strict metabolic regulation, are not subject to the normal feedback inhibition or activation mechanisms found in other cells. Moreover, our investigations and those of others support the hypothesis that a primary pathway for glucose metabolism is to phosphoenolpyruvic acid and then to oxalacetic acid with subsequent reduction to succinic acid.

Comparative metabolism of a new antileishmanial agent, allopurinol riboside, in the parasite and the host cell.

HPP-Rib is a potent antileishmanial agent, which has been useful in defining new and unusual purine metabolizing pathways in leishmaniae, in comparison with those in the host. The ribosyl linkage both in the parasite and in the host is resistant to cleavage. In the parasite there is a selective and marked conversion of HPP-Rib to HPP-Rib-5'-P and 4-APP ribonucleotides as well as incorporation into RNA, which does not occur in the host. These findings with HPP-Rib suggest a new chemotherapeutic approach which may be exploited in the treatment of leishmaniasis.