Fardos N. M. Naguib

Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus

A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).

Circadian rhythm of orotate phosphoribosyltransferase, pyrimidine nucleoside phosphorylases and dihydrouracil dehydrogenase in mouse liver: Possible relevance to chemotherapy with 5-fluoropyrimidines

In female mice (30-35 g) maintained in standardized conditions of 12 hr light (0600-1800 hr) alternating with 12 hr darkness (1800-0600 hr), food and water ad lib., there was a 24-hr cycle change (P < 0.0001, Cosinor analysis) in the activity of hepatic orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), uridine phosphorylase (UrdPase; EC 2.4.2.3), and dihydrouracil dehydrogenase (DHUDase; 1.3.1.2) but not thymidine phosphorylase (EC 2.4.2.4). The peaks of both OPRTase and UrdPase activities occurred in the activity span at around 18 and 15 hours after light onset (HALO) and the trough at 6 and 3 HALO, respectively, when samples were taken every 4 hr. Conversely, the peak of DHUDase occurred in the rest span at around 3 HALO and the trough at 15 HALO. The maximal enzyme activities (3146 +/- 172, 561 +/- 25, and 6.7 +/- 0.7 pmol/min/mg protein) was 210, 400 and 560% higher than the minimal activities (1507 +/- 172, 139 +/- 25, and 1.2 +/- 0.7 pmol/min/mg protein), for OPRTase UrdPase, and DHUDase, respectively. A circadian rhythm was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition there was a corresponding shift in UrdPase and DHUDase activities with a period of 1 hr difference in the time of maximum and minimum enzyme activities. OPRTase, on the other hand, showed little change after 4 weeks of adaptation under the reverse light cycle. The circadian rhythm of these key enzymes of pyrimidine metabolism, the interrelationship of their activities, and their role in the regulation of uridine bioavailability could be of particular significance in modulating the therapeutic regimens with 5-fluorinated pyrimidines.

6-Benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: Activities and selective toxicities

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-substituted purine nucleosides act as subversive substrates of T. gondii, but not the human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Herein, we report the testing of newly synthesized 6-benzylthioinosine analogues with various substituents on the phenyl ring of their benzyl group as subversive substrates of T. gondii adenosine kinases. The binding affinity of these compounds to T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents varied depending on the nature and position of the various substituents on the phenyl ring of their benzyl group. p-Cyano-6-benzylthioinosine and 2,4-dichloro-6-benzylthioinosine were the best ligands. In general, analogues with substitution at the para position of the phenyl ring were better ligands than those with the same substitutions at the meta or ortho position. The better binding of the para-substituted analogues is attributed to the combined effect of hydrophobic as well as van der Waals interactions. The 6-benzylthioinosine analogues were devoid of host-toxicity but all showed selective anti-toxoplasmic effect in cell culture and animal models. These results further confirm that toxoplasma adenosine kinase is an excellent target for chemotherapy and that 6-substituted purine nucleosides are potential selective antitoxoplasmic agents.

Uptake of Nitrobenzylthioinosine and Purine β-l-Nucleosides by Intracellular Toxoplasma gondii

ABSTRACT Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine {NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-β-d-ribofuranosylpurine}, an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological β-l-enantiomers of purine nucleosides, β-l-adenosine, β-l-deoxyadenosine, and β-l-guanosine. The β-l-pyrimidine nucleosides, β-l-uridine, β-l-cytidine, and β-l-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside β-l-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural β-l-nucleosides. β-d-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and β-l-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of β-d-adenosine, NBMPR, and purine β-l-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine β-l-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasites purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

Structure−Activity Relationships of 7-Deaza-6-benzylthioinosine Analogues as Ligands of Toxoplasma gondii Adenosine Kinase

Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza- p-cyano-6-benzylthioinosine (IC 50 = 5.3 microM) and 7-deaza- p-methoxy-6-benzylthioinosine (IC 50 = 4.6 microM), were evaluated in cell culture to delineate their selective toxicity.

Uridine phosphorylase inhibitors: chemical modification of benzyloxybenzyl-barbituric acid and its effects on urdpase inhibition.

5-(o-Benzyloxy)benzylbarbituric acid (6) and 5-(p-benzyloxy)benzylbarbituric acid (7) were prepared and their inhibitory activities compared to 5-(m-benzyloxy)-benzylbarbituric acid (BBB) a known, potent inhibitor of uridine phosphorylase (UrdPase). Compounds 6 and 7 were 18-fold and 51-fold less active, respectively, than BBB in inhibiting UrdPase. These data provide solid evidence that the 5-benzylbarbituric acids possessing meta substituents are the most active inhibitors. In addition, 2-thioBBB (11) was synthesized and it was shown to be as active an inhibitor as BBB.

Structure-activity relationships for the binding of ligands to xanthine or guanine phosphoribosyltransferase from Toxoplasma gondii

Abstract Preliminary characterization of Toxoplasma gondii phosphoribosyltransferase activity towards purine nucleobases indicates that there are at least two enzymes present in these parasites. One enzyme uses hypoxanthine, guanine, and xanthine as substrates, while a second enzyme uses only adenine. Furthermore, competition experiments using the four possible substrates suggest that there may be a third enzyme that uses xanthine. Therefore, sixty-eight purine analogues and thirteen related derivatives were evaluated as ligands of T. gondii phosphoribosyltransferase, using xanthine or guanine as substrates, by examining their ability to inhibit these reactions in vitro . Inhibition was quantified by determining apparent K i values for compounds that inhibited these activities by greater than 10% at a concentration of 0.9 mM. On the basis of these data, a structure-activity relationship for the binding of ligands to these enzymes was formulated using hypoxanthine (6-oxopurine) as a reference compound. It was concluded that the following structural features of purine analogues are required or strongly preferred for binding to both enzymes: (1) a pyrrole-type nitrogen (lactam form) at the 1-position; (2) a methine (CH), a pyridine type nitrogen (  N  ), or an exocyclic amino or oxo group at the 2-position; (3) no exocyclic substituents at the 3-position; (4) an exocyclic oxo or thio group in the one or thione tautomeric form at the 6-position; (5) a pyridine-type nitrogen (  N  ) or a methine group at the 7-position; (6) a methine group at the 8-position; (7) a pyrrole-type nitrogen or a carbon at the 9-position; and (8) no exocyclic substituents at the 9-position. These findings provide the basis for the rational design of additional ligands of hypoxanthine, guanine, and xanthine phosphoribosyltransferase activities in T. gondii .

Synthesis of [4-(hydroxy)tetrahydrofuran-2-yl]nucleosides as a novel class of uridine phosphorylase inhibitors

Abstract [4-(Hydroxy)tetrahydrofuran-2-yl]thymine was synthesized and found to be a potent inhibitor of uridine phosphorylase.

Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase.

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.

Modulation of plasma uridine concentration by 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase: relevance to chemotherapy.

Purpose: The purpose of this investigation was to evaluate the efficacy of oral 5-(phenylselenenyl)-acyclouridine (PSAU) in increasing endogenous plasma uridine concentration as well as its ability to improve the bioavailability of oral uridine. PSAU is a new potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. Methods: Oral PSAU was administered orally to mice alone or with uridine. The plasma levels of PSAU as well as uridine and its catabolites were measured using high-performance liquid chromatography and pharmacokinetic analysis was performed. Results: PSAU has an oral bioavailability of 100% and no PSAU metabolites were detected. PSAU has no apparent toxicity at high doses. Oral administration of PSAU at 30 and 120 mg/kg increased baseline concentration of endogenous plasma uridine (2.6 ± 0.7 μM) by 3.2- and 8.7-fold, respectively, and remained three- and six-fold higher, respectively, than the controls for over 8 h. PSAU, however, did not alter the concentration of endogenous plasma uracil. Co-administration of PSAU with uridine elevated the concentration of plasma uridine over that resulting from the administration of either alone, and reduced the peak plasma concentration (Cmax) and area under the curve (AUC) of plasma uracil. Co-administration of PSAU at 30 mg/kg and 120 mg/kg improved the low bioavailability of oral uridine (7.7%) administered at 1320 mg/kg by 4.8- and 4.2-fold, respectively, and reduced the AUC of plasma uracil from 1421 to 787 μmol/h · l and 273 μmol/h · l, respectively. Similar results were observed when PSAU was co-administered with lower doses of uridine. Oral PSAU at 30 mg/kg and 120 mg/kg improved the bioavailability of oral 330 mg/kg uridine by 5.2- and 8.9-fold, and that of oral 660 mg/kg uridine by 6.4- and 9.0-fold, respectively. However, the reduction in the AUC values of plasma uracil was less dramatic than that seen when the high dose of 1320 mg/kg uridine was used. Conclusion: The effectiveness of the PSAU plus uridine combination in elevating and sustaining high plasma uridine concentration may be useful to rescue or protect from host toxicity of various chemotherapeutic pyrimidine analogs as well as in the management of medical disorders that are remedied by administration of uridine.