The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology.

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.

2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies.

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.

Uridine phosphorylase from Escherichia coli. Kinetic properties and mechanism.

Type I and Type II uridine phosphorylases (uridine: orthophosphate ribosyltransferase EC 2.4.2.3) are distinguished by their pH optima (Krenitsky et al. (1965) J. Biol. Chem. 240, 1281-1286). A Type I enzyme was partially purified from Escherichia coli. The crossing pattern of the initial velocity analysis indicated that the catalytic mechanism involved the sequential addition of substrates to the enzyme. Product inhibition by uracil or by ribose 1-phosphate was linear competitive with uridine or with concentrations of phosphate below 3 mM. This indicated that the sequence of substrate addition was random rather than ordered. At concentrations of phosphate above 3 mM, product inhibition by uracil was complex. The random mechanism of this Type I enzyme contrasts with the ordered mechanism of a Type II enzyme from rat liver (Kraut, A. and Yamada, E.W. (1971) J. Biol. Chem. 246, 2021-2030).

Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases.

Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms.

Guanosine monophosphate synthetase from Ehrlich ascites cells. Multiple inhibition by pyrophosphate and nucleosides.

GMP synthetase (xanthosine-5'-phosphate: ammonia ligase (AMP-forming), EC 6.3.4.1) from Ehrlich ascites cells was found to be subject to multiple inhibition by its reaction product, PPi, and some analogs of adenosine. PPi and the nucleoside (N) inhibitors were also capable of individually inhibiting this enzyme. Under no conditions did the inhibition appear to be irreversible or "pseudoinactivating" in nature. The individual inhibition by PPi was competitive with respect to ATP (KI = 0.42 mM). Conversely, in the absence of PPi, the binding of N was noncompetitive with ATP, but shifted to a competitive pattern when PPi was present. Furthermore, with the inhibitors in concert, there was an apparent lowering of the KI values for both inhibitors. This data was consistent with either PPi functioning to tighten the binding of N at a noncatalytic site (positive cooperativity) or with PPi actually opening a second binding site for N in addition to the non-catalytic site. Although this study did not distinguish which of these events was occurring, it did reveal that the intensity of the effect of PPi appeared to be constant. That is, for various N inhibitors with a range of independently determined KI values from 26 to 1650 muM, the ratio of their KI values determined in the absence of PPi to the values determined in the presence of PPi was always 38 +/- 1.

Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species.

Ribonucleosides of some pyrazolo [3,4-d] pyrimidines have been shown to be potent anticoccidial agents. To investigate their interactions with adenosine kinase, this enzyme was purified by affinity chromatography from the sporulated oocysts of 3 avian coccidia, Eimeria tenella, E. acervulina and E. brunetti as well as from chicken liver. Comparative studies revealed several differences among the enzymes. Magnesium appeared not to be inhibitor of the E. tenella enzyme but did inhibit the enzymes from the other three sources. ATP in excess of the magnesium concentration strongly inhibited the E. brunetti enzyme but had only a small effect on the other enzymes. The chicken liver enzyme utilized a broader variety of triphosphate donors than did any of the enzymes from Eimeria species. ATP, dATP, GTP, dGTP and ITP was the best substrates. Studies with pyrazolo [3,4-d] pyrimidine nucleosides revealed two groups of enzymes with similar inhibitor specificities, the chicken liver and E. Acervulina vs. the E. tenella and E. brunetti enzyme. This grouping roughly correlates with the in vivo anticoccidial specificity of these compounds. Substrate specificity studies using two 4-substituted pyrazolo [3,4-d] pyrimidine ribonucleosides (ethylthio- and cinnamylthio-), which have shown potent anticoccidial activity in vivo, revealed that each served as a substrate for the enzymes from E. tenella and E. acervulina. The E. tenella enzyme was the more efficient at the phosphorylation of those compounds. However, only the ethylthio- compound was detectably phosphorylated by the enzyme from E. brunetti. In contrast to the inhibitor specificity, the substrate activities of these nucleosides do not correlate well with their in vivo anticoccidial activity.

Purine 2'-deoxy-2'-fluororibosides as antiinfluenza virus agents.

Twenty purine 2'-deoxy-2'-fluororibosides were synthesized by enzymic pentosyl transfer from 2'-deoxy-2'-fluorouridine. Each nucleoside analogue was assayed for cytotoxicity in uninfected Madin-Darby canine kidney cells and for their ability to suppress influenza A virus infections in these cells. The most potent antivirial activity was observed with analogues having an amino group in the 2-position of the purine moiety. All 2-unsubstituted analogues were less potent than their 2-amino counterparts. Furthermore, 2-methyl,2-methoxy, or 2-fluoro substitution obliterated antivirial activity. The most cytotoxic member of the series was the 2-fluoro-6-amino analogue (IC50 = 120 microM). 2'-Deoxy-2'-fluoroguanosine and those congeners readily converted to it by adenosine deaminase showed the most potent antivirial activity (IC50 = 15-23 microM). Little cytotoxicity was observed with this subgroup of analogues which renders them worthy of further investigation as potential antiinfluenza agents.

Nucleosides of azathioprine and thiamiprine as antiarthritics.

Azathioprine [Imuran; 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-1H-purine] is a widely used immunosuppressive and antiarthritic drug. For the sake of comparison, the riboside, the 2'-deoxyriboside, and the arabinoside of azathioprine and its 2-amino congener, thiamiprine, were prepared by an enzymatic method. In vitro, the cytotoxicities of these aglycons and their nucleosides were similar (ED50 = 0.8-2 microM), except for the arabinosides, which were nontoxic (ED50 greater than 100 microM). In vivo, their activities were compared in the rat adjuvant arthritis model. The ribosides and 2'-deoxyribosides were less potent than their corresponding aglycons. The safety indexes of these nucleosides were comparable to those of the corresponding aglycons except for the 2'-deoxyriboside of azathioprine, which had an appreciably lower safety index than did azathioprine. Both arabinosides were inactive and nontoxic. All of the aglycons tested (6-mercaptopurine, azathioprine, 6-thioguanine, and thiamiprine) were of similar potency. However, azathioprine had a more favorable therapeutic index than did 6-mercaptopurine. Similarly, thiamiprine was safer than was 6-thioguanine. In this model, the S-(1-methyl-4-nitro-1H-imidazol-5-yl) moiety imparted greater safety to these thiopurines by decreasing toxicity but not affecting potency.

Imidazo[4,5-c]pyridines (3-deazapurines) and their nucleosides as immunosuppressive and antiinflammatory agents.

A variety of imidazo[4,5-c]pyridines (3-deazapurines) were synthesized. With use of these aglycons as pentosyl acceptors, the corresponding ribonucleosides and 2'-deoxyribonucleosides were prepared by an enzymatic method involving transfer of the pentosyl moiety from appropriate pyrimidine nucleosides. With most of the imidazo[4,5-c]pyridines, the products obtained from the enzyme-catalyzed reactions were pentosylated exclusively in the 1-position. However, some 3-pentosylation occurred with aglycons that had H or N3 in the 4-position. In addition to the 2'-deoxy congener of the ribonucleoside of 4-amino-1H-imidazo[4,5-c]pyridine, the 5'-deoxy and 2',5'-dideoxy congeners were synthesized. All of the aglycons and their nucleosides were tested for toxicity to mammalian cells in culture. None were markedly cytotoxic. These compounds were also evaluated for their ability to inhibit lymphocyte-mediated cytolysis in vitro. 3-Deazaadenosine (23) and its 2'-deoxy congener (38) were the most potent inhibitors (ED50 = 20 microM). In addition to these two in vitro tests, in vivo inhibition of the inflammatory response in the rat carregeenan pleurisy model was determined. 3-Deazaadenosine (23) was the most potent compound (ED50 = 3 mg/kg) in this in vivo test.

Di- and triester prodrugs of the varicella-zoster antiviral agent 6-methoxypurine arabinoside.

6-Methoxypurine arabinoside (9-beta-D-arabinofuranosyl-6-methoxy-9H-purine, 1) has potent and selective activity against varicella-zoster virus in vitro. An unfavourable metabolic profile observed with oral dosing in the rat led to the preparation of a variety of 2',3',5'-triesters (2a-n) and several 2',3'-, 2',5'-, and 3',5'-diesters of this arabinoside (3a-n, 4a-f, and 5a-j, respectively). The compounds were evaluated as prodrugs by measuring the urinary levels of 1 in rat urine after oral dosing. With the exception of triacetate 2a, the triesters failed to significantly enhance bioavailability. Administration of compound 2a resulted in a 3-fold increase in systemic availability of 1, possibly because of its increased water solubility (1.6 times more soluble than 1) and only slightly increased relative log P value (1.93 vs 0.50 for 1). The longer chain aliphatic triesters and aromatic triesters had lower water solubilities and increased lipophilic partitioning. These factors might account for the lower systemic bioavailability of these compounds. In contrast, the diesters, especially the aliphatic diesters, showed significantly improved systemic availability. This might be a consequence of the higher aqueous solubilities and enhanced partition coefficients seen with these compounds. 2',3'-Diacetate 3a showed the best combination of high systemic availability and water solubility of all the prodrugs of 1.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 2. Synthesis and activity of some nucleosides of 4-(alkylamino)-1H-pyrazolo[3, 4-d]pyrimidines.

A series of 4-(alkylamino)-1-beta-D-ribofuranosyl-1H-pyrazolo[3, 4-d]pyrimidines was synthesized by enzymatic and chemical methods. On the basis of the previous finding that 4-(alkylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidines were effective anticoccidial agents, this series was examined for efficacy against Eimera tenella in chicks. The most active anticoccidial agent in the present study was the 4-cyclopentylamino derivative (8), which cleared chicks of the parasite at 200 ppm in the diet. Some members of this series were toxic to embryonic chick liver cells, mouse cells, and human cells in vitro. The 4-diethylamino derivative (16), which was not toxic in vitro, appeared to be toxic in chicks.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 1. Synthesis and activity of some nucleosides of purines and 4-(alkylthio)pyrazolo[3,4-d]pyrimidines.

The finding that 6-(methylthio)-9-beta-D-ribofuranosyl-9H-purine (6) was more toxic to the avian coccidium, Eimeria tenella, than to embryonic chick liver host cells in vitro prompted the synthesis and testing of analogues of this compound. It was revealed that the beta-D-ribofuranosyl moiety was an important structural feature and that several types of 2-substituents in the purine ring decreased efficacy, as did 3-deaza and 8-aza ring modifications of 6. In contrast, the pyrazolo[3,4-d]pyrimidine analogue of 6 (24) was an order of magnitude more active. Moreover, this analogue was 24-fold less toxic to the host cells than was 6. A series of 4-(alkylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidines was prepared from 4-mercapto-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (23) and various alkyl halides. The most effective compound in this series in vivo, 4-(ethylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (25), cleared chicks of the parasite at 50 ppm in the diet and was much less toxic than was 24.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 3. Synthesis and activity of some nucleosides of 4-[(arylalkenyl)thio]pyrazolo[3,4-d]pyrimidines.

Ribonucleosides of 4-(alkylthio)-1H-pyrazolo[3,4-d]pyrimidines have been shown to be useful anticoccidial agents [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32. Rideout, J. L.; Krenitsky, T. A.; Elion, G. B. U.S. Patent 4299 283, 1981]. In that study, the unsaturated 4-allylthio and 4-crotylthio derivatives (19 and 20) were shown to be more active in vivo against Eimeria tenella than their saturated congeners; therefore, some unsaturated (arylalkyl)thio derivatives were synthesized and investigated as anticoccidial agents. The novel compounds in this study (2 to 18) were prepared by the alkylation of 4-mercapto-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (1), which was prepared by an enzymatic method. The (E)-4-cinnamylthio derivative (2) and the 5'-monophosphate (18) were the most active compounds against E. tenella in vivo. None of the analogues with substituents in the aryl moiety (3 to 13) was more active than 2 in vivo. The geometry about the double bond was important, since the (Z)-4-cinnamylthio derivative (14) was inactive both in vitro and in vivo. The 4-(3-phenylpropynyl)thio and 4-(5-phenyl-2,4-pentadienyl)thio derivatives (15 and 16) were at least as active as 2 in vitro; however, they were less active than 2 in vivo. Compound 2 was effective in vivo against E. tenella, E. necatrix, E. maxima, and E. brunetti; these species of Eimeria were controlled when 2 was given in the diet at levels upt to 100 ppm. Infections in vivo due to E. acervulina were controlled by 2 only at about 800 ppm. The broad spectrum of anticoccidial activity shown by 2 represents a significant improvement over the activities reported for related compounds [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32].

3'-Amino-2',3'-dideoxyribonucleosides of some pyrimidines: synthesis and biological activities.

3'-Amino-2',3'-dideoxyribonucleosides of thymine, uracil, and 5-iodouracil (1-3) were synthesized from the corresponding 2'-deoxyribonucleosides via the threo-3'-chloro and the erythro-3'-azido derivatives. Corresponding aminonucleosides of 5-bromouracil, 5-chlorouracil, and 5-fluorouracil (4-6) were synthesized enzymatically with 3'-amino-2',3'-dideoxythymidine as the aminopentosyl donor and thymidine phosphorylase (EC 2.4.2.4) as the catalyst. 3'-Amino-2',3'-dideoxycytidine (7) was synthesized by amination of the 3'-azido precursor of 3'-amino-2',3'-dideoxyuridine. The biological activity of 3'-amino-2',3'-dideoxy-5-fluorouridine (6) was notable among this group of aminonucleosides. It had an ED50 of 10 microM against adenovirus and was not appreciably cytotoxic to mammalian cells in culture. It also had activity against some Gram-positive bacteria but not against a variety of Gram-negative bacteria. The other aminonucleosides (1-5 and 7) lacked or exhibited weak antiviral and antibacterial activities. The only compounds in this group that were appreciably toxic to mammalian cells in culture were the thymidine and deoxycytidine analogues (1 and 7).

Novel 6-alkoxypurine 2',3'-dideoxynucleosides as inhibitors of the cytopathic effect of the human immunodeficiency virus.

Twenty-one 6-alkoxypurine 2',3'-dideoxynucleosides were enzymatically synthesized with nucleoside phosphorylases purified from E. coli. Eighteen analogs exhibited anti-HIV-1 activity in MT4 cells. Two analogs, 6-(hexyloxy)-(17) and 6-(heptyloxy)-(18) purine 2',3'-dideoxynucleoside, were as potent as 2',3'-dideoxyinosine (ddI, didanosine, Videx). Although the antiviral activities of 17 and 18 were equivalent, 18 was more cytotoxic. Analogs containing less than four carbons in the 6-alkoxypurine substituent exhibited weak anti-HIV-1 activity. Analogs containing more than seven carbons in the 6-alkoxypurine substituent were too cytotoxic to be effectively evaluated for antiviral activity. Several 6-alkoxypurine 2',3'-dideoxynucleosides were evaluated for substrate activity with calf intestinal adenosine deaminase (ADA). Increasing the carbon chain length of the 6-alkoxypurine substituent decreased the rate of dealkoxylation. The best substrate in this series was 6-methoxypurine 2',3'-dideoxynucleaside (1); however, the rate of dealkoxylation of 100 microM 1 was 0.17% of the rate of deamination of 100 microM 2',3'-dideoxyadenosine. Compound 17, the most potent anti-HIV-1 analog, was not a substrate for ADA. EHNA (erthro-9-(2-hydroxy-3-nonyl)adenine), a potent inhibitor of ADA, had little effect on the antiviral activities of 17 and ddI. In contrast, coformycin, a potent inhibitor of both ADA and AMP deaminase, dramatically decreased the antiviral activity of 17, but not the antiviral activity of ddI. Thus, AMP deaminase appeared to be involved in the anabolism of 17. The pharmacokinetic profile of 17, the most promising analog in this series, was determined in the rat. At least seventeen metabolites of 17, including ddI, were detected in plasma samples. This analog also had poor oral bioavailability.

Human and bovine xanthine oxidases. Inhibition studies with oxipurinol.

Oxipurinol inhibited human xanthine oxidase and bovine xanthine oxidases by very similar mechanisms. It bound to an electronically reduced form of human xanthine oxidase in a manner similar to that previously discerned from its interactions with the bovine enzyme [review article: Spector, Biochem. Pharmac. 26, 355 (1977)]. Xanthine was a good source for the reducing equivalents because it did not compete with oxipurinol for binding to reduced enzyme. The inhibition of the rate of urate production progressively increased with time. Studies of the effect of the concentration of oxipurinol on the rate constant of the development of this inhibition revealed that a complex was rapidly formed between oxipurinol and reduced bovine or human xanthine oxidases (KD of about 8 microM). At 37 degrees these complexes were converted to stable complexes at a maximum rate of about 1.6 min-1. The rate constant was highly temperature dependent with an energy of activation of 30 kcal/mole (cf. 13 kcal/mole for the energy of activation for catalysis). These data support the earlier conclusions that the formation of stable complexes probably reflects a massive rearrangement of the initial complexes. The isolated oxipurinol-xanthine oxidase complexes spontaneously reverted to active enzyme with a rate constant of 0.02 min-1 at 37 degrees. The energy of activation for the "reactivation" was similar to that for the formation of the stable complexes. The rates of "reactivation" could be stimulated by high concentrations of xanthine: 2.4-fold at 50 microM and 3.4-fold at 100 microM. The constant for the overall inhibition by oxipurinol was approximately 100 nM with both enzymes.

Trypanosoma cruzi adenine nucleoside phosphorylase. Purification and substrate specificity.

An adenine nucleoside phosphorylase has been partially purified from extracts of epimastigotes of the Peru strain of Trypanosoma cruzi, the causative agent of Chagas' disease. The purification procedure separated this enzyme from the three other nucleoside-cleaving enzymes found in extracts. The adenine nucleoside phosphorylase, which efficiently cleaved 5'-deoxy-5'-methylthioadenosine (MTA), had a particle weight of 68,000 and exhibited a broad pH optimum between pH 6 and 8. In addition to MTA, the purified enzyme cleaved and synthesized adenosine and 2'-deoxyadenosine with high efficiency. This contrasts to the enzyme from S-180 cells which has been reported to cleave adenosine poorly and not to cleave 2'-deoxyadenosine. Several observations suggested that the three substrates, MTA, adenosine and 2'-deoxyadenosine, use a common catalytic site: (a) all served as alternate-substrate inhibitors exhibiting mutually competitive inhibition with Ki values equivalent to their respective Km values, (b) 5'-chloroformycin A exhibited a competitive Ki value of 4 microM with each nucleoside substrate, and (c) the Km value of phosphate derived from initial velocity studies (180 +/- 20 microM) was independent of the nucleoside substrate. Substrate specificity studies in both the synthesis and cleavage direction indicated that the enzyme had a broad specificity for bases and nucleosides. For the synthesis of nucleosides, the enzyme demonstrated a preference for an amino group in the position equivalent to the 6 position of purine. Compounds containing a hydroxyl group in this position were not substrates. Although a hydrogen or methyl group could substitute for a 6-amino group, a marked decrease in substrate efficiency was observed with these compounds. Alterations in the purine ring led to decreases in the maximal velocity values as evidenced by the substrate or nonsubstrate properties of 1-, 3-, and 7-deazaadenine and 4-aminopyrazolo[3,4-d]pyrimidine. The Km values for 5-methylthioribose 1-phosphate, ribose 1-phosphate and 2'-deoxyribose 1-phosphate with adenine serving as acceptor were 21, 150 and 370 microM. For nucleoside cleavage, the T. cruzi enzyme catalyzed the phosphorolysis of a variety of 5'-substituted adenine-containing nucleosides including those possessing 5'-hydrogen-, hydroxyl-, halogeno-, alkylthio-, amino- and azido-moieties. Inclusion of an ionized group in the 5'-position, such as 5'-carboxy-5'-deoxyadenosine or AMP, precluded substrate activity. 3'-Deoxyadenosine, arabinosyladenine and alpha-adenosine did not serve as substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase.

Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.

Inhibition of uridine phosphorylase from Escherichia coli by benzylacyclouridines.

The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli. Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-[(2'-hydroxy-1'-hydroxymethyl)methyl]urac il) with a Ki value of 0.15 microM. The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources [Niedzwicki et al., Biochem. Pharmac. 31, 1857 (1982) and Naguib et al., manuscript in preparation] indicating that the structure of the active site of uridine phosphorylase from E. coli may resemble that of the mammalian enzyme.

Antigiardial activity of guanine arabinoside. Mechanism studies.

Guanine arabinoside (araG) inhibited the in vitro growth of Giardia lamblia WB with an ED50 value of 4 microM. The inhibition was prevented completely by 2'-deoxyguanosine, prevented partially by guanine and guanosine, and not prevented by adenine, adenosine or 2'-deoxyadenosine. Extracts of G. lamblia grown in the presence of [8-3H]araG contained radiolabeled araGMP, araGDP and araGTP. The formation of araGTP during the exponential phase of cell growth increased with time and was dependent upon the araG concentration. AraG was incorporated into G. lamblia DNA in a time-dependent manner at a ratio of 1 araG for each 27 2'-deoxyguanosine residues. Short-term exposure of growing cultures to araG was inhibitory to DNA synthesis but not to RNA or protein synthesis. Over an extended period, synthesis of all three macromolecules was depressed. Attempts to measure araG phosphorylation by cell-free extracts of G. lamblia under a variety of nucleoside kinase and nucleoside phosphotransferase assay conditions were unsuccessful. In an attempt to understand further the action of araG, the metabolic pathways of guanine, guanosine and 2'-deoxyguanosine were delineated in detail. The presence of araG did not appear to cause any major alterations in the metabolism of these compounds; however, it was accompanied by a 3- to 4-fold increase in the endogenous pools of ATP and GTP.