Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine.

Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo=0.002±0.0002 mM, KATP=0.05±0.008 mM, and Vmax=920±35 µmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.

Potent combination therapy for human breast tumors with high doses of 5-fluorouracil: remission and lack of host toxicity.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host toxicity and enhancing its chemotherapeutic efficacy against human breast tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UP, EC 2.4.2.3), the enzyme responsible for uridine catabolism. METHODS: SCID mice bearing MDA-MB-468 and MCF-7 human breast tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24, and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg), or their combination was administered orally two or 4 h after FUra injection. Another four administrations of PTAU plus uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. RESULTS: Administration of the maximum tolerated dose (50 mg/kg) of 5-fluorouracil (FUra) to SCID mice bearing human breast MDA-MB-468 and MCF-7 adenocarcinoma tumor xenografts reduced tumor weight by 59 and 61%, respectively. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not rescue from FUra host toxicity as all the mice died. Administration of 120 mg/kg PTAU resulted in partial rescue from this lethal dose of FUra as 38% of inoculated mice survived and the tumor weights were reduced by approximately 67%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra. All of the mice survived, and MDA-MB-468 and MCF-7 tumor weights were reduced by 97% and total remission, respectively. Doubling the FUra treatment dose to 400 mg/kg in the MDA-MB-468 inoculated mice, with the administration of the adjuvant combination treatment of PTAU plus uridine, was unsuccessful in rescuing from FUra toxicity as all the mice died. Lowering the dose of FUra to 300 mg/kg, under the same conditions, resulted in 67% mice survival, and the MCF-7 tumor weights were reduced by 100%. Treatment with uridine alone did not protect from FUra toxicity at 200, 300, and 400 mg/kg as all of the mice died. At the higher dose of 300 and 400 mg/kg FUra, PTAU alone had no rescuing effect. There was no significant difference between MDA-MB-468 and MCF-7 in their response to the different regimens employed in this study in spite of the fact that MDA-MB-468 is estrogen receptor negative while MCF-7 is estrogen receptor positive. CONCLUSIONS: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combination treatment in raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al. in Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the massive doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side effects associated with such doses of uridine. The combination may also allow the escalation of FUra doses for better chemotherapeutic efficacy against human breast carcinoma, with the possibility of avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

Carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase: biological 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. Several 6-benzylthioinosines have already been identified as subversive substrates of the T. gondii but not human adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not its host. In the present study, we report the testing of the metabolism of several carbocyclic 6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the carbocyclic 6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the carbocyclic 6-benzylthioinosine analogues showed a selective anti-toxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. These results indicate that the oxygen atom of the sugar is not critical for substrate binding. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were carbocyclic 6-(p-methylbenzylthio)inosine (IC(50)=11.9 microM), carbocyclic 6-(p-methoxybenzylthio)inosine (IC(50)=12.1 microM), and carbocyclic 6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=12.8 microM). These compounds have about a 1.5-fold better efficacy relative to their corresponding 6-benzylthioinosine analogues (Rais et al., Biochem Pharmacol 2005;69:1409-19 [29]). The results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that carbocyclic 6-benzylthioinosines are potential anti-toxoplasmic agents.

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.

7-Deaza-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-benzylthioinosines act as subversive substrates of T. gondii, but not human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Moreover, 7-deazaadenosine (tubercidin) was shown to be an excellent ligand of T. gondii adenosine kinase. Therefore, we synthesized 7-deaza-6-benzylthioinosine, and analogues with various substitutions at their phenyl ring, to increase the binding affinity of the 6-benzylthioinosines to T. gondii adenosine kinase. Indeed, the 7-deaza-6-benzylthioinosine analogues were better ligands of T. gondii adenosine kinase than the parent compounds, 6-benzylthioinosine and 7-deazainosine. Herein, we report the testing of the metabolism of these newly synthesized 7-deaza-6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the 7-deaza-6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the 7-deaza-6-benzylthioinosine analogues showed a selective antitoxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were 7-deaza-6-(p-methoxybenzylthio)inosine (IC(50)=4.6 microM), 7-deaza-6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=5.0 microM), and 7-deaza-6-(p-cyanobenzylthio)inosine (IC(50)=5.3 microM). These results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that 7-deaza-6-benzylthioinosines are potential antitoxoplasmic agents.

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.

Synthesis, biological evaluation and molecular modeling studies of N6-benzyladenosine analogues as potential anti-toxoplasma agents.

Toxoplasma gondii is an opportunistic pathogen responsible for toxoplasmosis. T. gondii is a purine auxotroph incapable of de novo purine biosynthesis and depends on salvage pathways for its purine requirements. Adenosine kinase (EC.2.7.1.20) is the major enzyme in the salvage of purines in these parasites. 6-Benzylthioinosine and analogues were established as "subversive substrates" for the T. gondii, but not for the human adenosine kinase. Therefore, these compounds act as selective anti-toxoplasma agents. In the present study, a series of N(6)-benzyladenosine analogues were synthesized from 6-chloropurine riboside with substituted benzylamines via solution phase parallel synthesis. These N(6)-benzyladenosine analogues were evaluated for their binding affinity to purified T. gondii adenosine kinase. Furthermore, the anti-toxoplasma efficacy and host toxicity of these compounds were tested in cell culture. Certain substituents on the aromatic ring improved binding affinity to T. gondii adenosine kinase when compared to the unsubstituted N(6)-benzyladenosine. Similarly, varying the type and position of the substituents on the aromatic ring led to different degrees of potency and selectivity as anti-toxoplasma agents. Among the synthesized analogues, N(6)-(2,4-dimethoxybenzyl)adenosine exhibited the most favorable anti-toxoplasma activity without host toxicity. The binding mode of the synthesized N(6)-benzyladenosine analogues were characterized to illustrate the role of additional hydrophobic effect and van der Waals interaction within an active site of T. gondii adenosine kinase by induced fit molecular modeling.

Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(Phenylthio)acyclouridine, a uridine phosphorylase inhibitor.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host-toxicity and enhancing its chemotherapeutic efficacy against human colon tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. METHODS: SCID mice bearing human colon DLD-1 or HCT-15 tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24 and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg) or their combination was administered orally 2 or 4 h after FUra injection. Another four administrations of PTAU+uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. RESULTS: Administration of the maximum tolerated dose (50 mg/kg) of FUra reduced DLD-1 and HCT-15 tumor weights by 48 and 59%, respectively, at day 38 post implantation. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not alleviate FUra host-toxicity as all the mice died. Administration of 120 mg/kg PTAUresulted in partial rescue from this lethal dose of FUra as 63% of mice survived and tumor weights were reduced by approximately 60%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra as 100% of the mice survived and tumor weights were reduced by 81-82%. Delaying the administration of the combination of PTAU plus uridine to 4 h post FUra treatment was less effective in rescuing from FUra toxicity as only 88% of the mice survived and tumor weights were reduced by only 62%. Administration of PTAU alone, under the same conditions, resulted in a 38% survival rate while the tumor weights were reduced by 47%. Treatment with uridine alone did not protect from FUra toxicity at the dose of 200 mg/kg as all mice died. At the higher dose of 300 mg/kg FUra, neither uridine nor PTAU alone, administered 2 h following the treatment with FUra, had any rescuing effect. On the other hand, the use of the PTAU plus uridine combination reduced the tumor weight by 79%, although this reduction in the tumor weight was accompanied by 37% mortality. There was no significant difference between DLD-1 and HCT-15 in their response to the different regimens employed in this study despite the fact that the tumors have different levels of UrdPase. CONCLUSIONS: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host-toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combinationin raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al., Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the large doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side-effects associated with such doses of uridine. This combination may also allow for the escalation of FUra doses for better chemotherapeutic efficacy against human colon carcinoma while avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

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.

5-(Phenylthio)acyclouridine: a powerful enhancer of oral uridine bioavailability: relevance to chemotherapy with 5-fluorouracil and other uridine rescue regimens.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the pharmacokinetics and bioavailability of oral uridine. PTAU is a 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. PTAU is fully absorbed after oral administration with 100% oral bioavailability. METHODS: Uridine (330, 660 or 1320 mg/kg) and/or PTAU (30, 45, 60, 120, 240 or 480 mg/kg) were orally administered to mice. The plasma levels of uridine, its catabolite uracil, and PTAU were measured using HPLC, and pharmacokinetic analysis was performed. RESULTS: Oral PTAU up to 480 mg/kg per day is not toxic to mice. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg has a prolonged plasma half-life of 2-3 h, and peak plasma PTAU concentrations (C(max)) of 41, 51, 74, 126 and 161 microM with AUCs of 70, 99, 122, 173 and 225 micromol h/l, respectively. Coadministration of uridine with PTAU did not have a significant effect on the pharmacokinetic parameters of plasma PTAU at any of the doses tested. Coadministration of PTAU (30, 45, 60 and 120 or 240 mg/kg) with uridine (330, 660 or 1320 mg/kg) elevated the concentration of plasma uridine over that following the same dose of uridine alone, a result of reduced metabolic clearance of uridine as evidenced by decreased plasma exposure (C(max) and AUC) to uracil. Plasma uridine was elevated with the increase of uridine dose at each PTAU dose tested and no plateau was reached. Coadministration of PTAU at 30, 45, 60, 120 and 240 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9-, 9.9-, 11.7- and 12.5-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol h/l) by 5.7-, 6.8-, 8.2-, 6.3-, and 6.9-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.7-, 2.4-, 2.6-, 5.2- and 4.3- fold, and that of 660 mg/kg uridine by 2.3-, 2.7-, 3.3-, 4.6- and 6.7-fold, respectively. CONCLUSION: The excellent pharmacokinetic properties of PTAU, and its extraordinary effectiveness in improving the oral bioavailability of uridine, could be useful to rescue or protect from host toxicities of 5-fluorouracil and various chemotherapeutic pyrimidine analogues used in the treatment of cancer and AIDS, as well as in the management of medical disorders that are remedied by the administration of uridine including CNS disorders (e.g. Huntington's disease, bipolar disorder), liver diseases, diabetic neuropathy, cardiac damage, various autoimmune diseases, and transplant rejection.

Synthesis, biological activity and molecular modeling of 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase.

Toxoplasma gondii is the most common cause of secondary CNS infections in immunocompromised persons such as AIDS patients. The major route of adenosine metabolism in T. gondii is direct phosphorylation to adenosine 5'-monophosphate (AMP) catalyzed by the enzyme adenosine kinase (EC 2.7.1.20). Adenosine kinase in T. gondii is significantly more active than any other purine salvage enzyme in this parasite and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Subversive substrates of T. gondii,but not the human, adenosine kinase are preferentially metabolized to their monophosphorylated forms and become selectively toxic to the parasites but not their host. 6-Benzylthioinosine (BTI) was identified as an excellent subversive substrate of T. gondii adenosine kinase. Herein, we report the synthesis of new analogues of BTI as subversive substrates for T. gondii adenosine kinase. These new subversive substrates were synthesized starting from tribenzoyl protected d-ribose. To accomplish the lead optimization process, a divergent and focused combinatorial library was synthesized using a polymer-supported trityl group at the 5'-position. The combinatorial library of 20 compounds gave several compounds more active than BTI. Structure-activity relationship studies showed that substitution at the para position plays a crucial role. To investigate the reasons for this discrimination, substrates with different substituents at the para position were studied by molecular modeling using Monte Carlo Conformational Search followed by energy minimization of the enzyme-ligand complex.

Uptake of nitrobenzylthioinosine and purine beta-L-nucleosides by intracellular Toxoplasma gondii.

Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine [NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-beta-D-ribofuranosylpurine], an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological beta-L-enantiomers of purine nucleosides, beta-L-adenosine, beta-L-deoxyadenosine, and beta-L-guanosine. The beta-L-pyrimidine nucleosides, beta-L-uridine, beta-L-cytidine, and beta-L-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside beta-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 beta-L-nucleosides. beta-D-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and beta-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 beta-D-adenosine, NBMPR, and purine beta-L-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine beta-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 parasite's 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.