RESUMO
BACKGROUND: The use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation. PATIENTS AND METHODS: A first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m(2)) and escalating virus in the fourth (10(11)-10(12) viral particles, VP). RESULTS: All 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy. CONCLUSIONS: This first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted. CLINICALTRIALSGOV IDENTIFIER: NCT01310179.
Assuntos
Escherichia coli/enzimologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Neoplasias/genética , Neoplasias/terapia , Purina-Núcleosídeo Fosforilase/administração & dosagem , Vidarabina/análogos & derivados , Adenoviridae/genética , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/patologia , Prognóstico , Purina-Núcleosídeo Fosforilase/genética , Células Tumorais Cultivadas , Vidarabina/uso terapêuticoRESUMO
The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-ß-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.
Assuntos
Adenina Fosforribosiltransferase/metabolismo , Escherichia coli/enzimologia , Pró-Fármacos/farmacologia , Purina-Núcleosídeo Fosforilase/metabolismo , Animais , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Terapia Genética , Vetores Genéticos/genética , Humanos , Camundongos , Pró-Fármacos/uso terapêutico , Nucleosídeos de Purina/metabolismo , Transplante Heterólogo , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/metabolismoRESUMO
A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5-position have been synthesised These compounds bear a 5'-heterocycle such as triazole or a imidazole with a two carbon chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl purine nucleosides, their corresponding alpha-linker nucleosides with L-xylose and L-lyxose were also synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP.
Assuntos
Adenina/análogos & derivados , Terapia Genética/métodos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Nucleosídeos/síntese química , Pró-Fármacos/farmacologia , Purinas/química , Adenina/farmacologia , Antineoplásicos/farmacologia , Carbono/química , Escherichia coli/enzimologia , Humanos , Modelos Químicos , Mutação , Nucleosídeos/química , Pró-Fármacos/química , Nucleosídeos de Purina/química , Purina-Núcleosídeo Fosforilase/química , Especificidade por Substrato , Xilose/químicaRESUMO
During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.
Assuntos
Escherichia coli/enzimologia , Terapia Genética , Neoplasias/terapia , Pró-Fármacos/farmacocinética , Purina-Núcleosídeo Fosforilase/metabolismo , Animais , Biotransformação , Flucitosina/farmacocinética , Ganciclovir/farmacocinética , Camundongos , Camundongos Nus , Purina-Núcleosídeo Fosforilase/genética , Simplexvirus/enzimologia , Timidina Quinase/genéticaRESUMO
Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.
Assuntos
Adenina/análogos & derivados , Purinas/metabolismo , Células 3T3 , Adenina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cicloeximida/farmacologia , DNA/efeitos dos fármacos , DNA/metabolismo , Fluoruracila/farmacologia , Humanos , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA/efeitos dos fármacos , RNA/metabolismoRESUMO
The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.
Assuntos
5'-Nucleotidase/metabolismo , Desoxicitidina Quinase/metabolismo , Desoxiguanosina/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Células Cultivadas/enzimologia , Células Cultivadas/virologia , Citomegalovirus/efeitos dos fármacos , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Desoxiguanosina/farmacologia , Patos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Ácido Micofenólico/farmacologia , Nucleosídeos/farmacologia , Fosforilação/efeitos dos fármacos , Estereoisomerismo , Especificidade por SubstratoRESUMO
The polyamines putrescine, spermine, and spermidine, present in all living cells, have been implicated in the replication of some herpesviruses and retroviruses, and elevated levels of these polyamines have been found in the lymphocytes of patients infected with HIV-1. We have examined the effect of HIV-1 infection on polyamine pools in cell culture. HIV-1 did not significantly affect the polyamine pools in CEM cells. Consistent with this observation, inhibitors of the two key enzymes of this pathway, ornithine decarboxylase and S-adenosylmethionine decarboxylase, did not prevent viral-induced cytopathic effects (CPE) in this cell line. Our results indicate that inhibitors of this pathway will not be therapeutically useful in the treatment of AIDS.
Assuntos
HIV-1/crescimento & desenvolvimento , Poliaminas/análise , Linfócitos T/virologia , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Humanos , Inibidores da Ornitina Descarboxilase , Putrescina/análise , S-Adenosilmetionina/antagonistas & inibidores , Espermidina/análise , Espermina/análiseRESUMO
Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.
Assuntos
Comunicação Celular , Escherichia coli/enzimologia , Purina-Núcleosídeo Fosforilase/farmacologia , Purinas/farmacologia , Marcadores de Afinidade , Animais , Morte Celular , Divisão Celular , Meios de Cultura , Humanos , Camundongos , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Purina/farmacologia , Purina-Núcleosídeo Fosforilase/metabolismo , Purinas/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Células Tumorais CultivadasRESUMO
For 3 consecutive days, the nucleoside cordycepin (3'-deoxyadenosine) was administered as 1-hr iv infusions (0, 1, 4, 8, 10, or 20 mg/kg/day) to dogs. These doses were given 1 hr after a bolus iv injection (0.25 mg/kg/day) of 2'-deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase. The hypothesis was that dCF would affect the toxicity of cordycepin. Plasma adenosine deaminase activity was strongly inhibited during the dose period and for 5 days following the final dose of dCF. Dogs given cordycepin alone showed no drug-related toxicities. In dogs given only dCF, drug-related toxicity to lymphoid tissue (lymphopenia and thymus lymphoid depletion), thrombocytopenia, and decreases in food consumption were observed. Cordycepin in combination with dCF produced symptoms associated with severe gastrointestinal toxicity (decreased body weights, emesis, diarrhea, decreased food consumption, and necrosis of the gastrointestinal tract) and bone marrow toxicity (lymphopenia, thrombocytopenia, and depletion of hematopoietic cells). The gastrointestinal tract and bone marrow were sites associated with dose-limiting toxicities. In surviving dogs, most of the effects were reversible by Day 30. The maximum tolerated dose of cordycepin administered in combination with dCF was 8 mg/kg/day (160 mg/m2/day) given daily for 3 days.
Assuntos
Adenosina Desaminase/sangue , Antibióticos Antineoplásicos/toxicidade , Antineoplásicos/toxicidade , Desoxiadenosinas/toxicidade , Pentostatina/toxicidade , Inibidores de Adenosina Desaminase , Animais , Antibióticos Antineoplásicos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Desoxiadenosinas/administração & dosagem , Cães , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Inibidores Enzimáticos/toxicidade , Gastroenteropatias/induzido quimicamente , Infusões Intravenosas , Injeções Intravenosas , Contagem de Leucócitos/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/patologia , Pentostatina/administração & dosagem , Contagem de Plaquetas/efeitos dos fármacos , Trombocitopenia/induzido quimicamenteRESUMO
We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Terapia Genética/métodos , Glioma/tratamento farmacológico , Pró-Fármacos/farmacologia , Purina-Núcleosídeo Fosforilase/fisiologia , Animais , Antimetabólitos Antineoplásicos/toxicidade , Escherichia coli/enzimologia , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Nucleosídeos de Purina/uso terapêutico , Nucleosídeos de Purina/toxicidade , Purina-Núcleosídeo Fosforilase/genética , Retroviridae/genética , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/uso terapêutico , Fosfato de Vidarabina/toxicidadeRESUMO
Inefficiency of gene delivery, together with inadequate bystander killing, represent two major hurdles in the development of a toxin-mediated gene therapy for human malignancy. The product of the Escherischia coli DeoD gene (purine nucleoside phosphorylase, PNP) differs from the mammalian enzyme in its substrate specificity and is capable of catalyzing the conversion of several non-toxic deoxyadenosine analogs to highly toxic adenine analogs. We have found that expression of E. coli PNP in < 1% of a human colonic carcinoma cell line leads to the death of virtually all bystander cells after treatment with 6-methyl-purine-2'-deoxyribonucleoside, a deoxyadenosine analog that is a substrate for E. coli PNP but not human PNP. Minimal toxicity was observed in non-transfected or E. coli LacZ transfected cells that were treated with this compound. These results establish a rational approach to achieve significant bystander killing, even after gene transfer to only a small fraction of tumor cells.
Assuntos
Neoplasias do Colo/terapia , Escherichia coli/genética , Terapia Genética , Sequência de Bases , Morte Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Primers do DNA/genética , Técnicas de Transferência de Genes , Genes Bacterianos , Humanos , Óperon Lac , Dados de Sequência Molecular , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Purinas/biossíntese , Purinas/toxicidade , Transfecção , Células Tumorais CultivadasRESUMO
6-Thio-2'-deoxyguanosine (T-dGuo) has been reported to be both phosphorylated by deoxycytidine kinase and converted to 6-thioguanine by purine nucleoside phosphorylase (PNP). Combination of T-dGuo with an inhibitor of PNP would be expected to generate the 5'-triphosphate of T-dGuo and limit or prevent the formation of 6-thioguanosine triphosphate. Because the incorporation of 6-thioguanine into DNA is believed to be primarily responsible for the antitumor activity of the thiopurines, this treatment might result in enhanced activity against certain tumors, particularly those of T-cell origin. We have evaluated the metabolic basis of this strategy by examining the effects of 9-benzyl-9-deazaguanine (BDG), a potent inhibitor of PNP, on the metabolism of T-dGuo in CEM cells. The concentration of T-dGuo required to inhibit cell growth by 50% was approximately 50-fold greater in the presence of 8.0 microM BDG than in its absence. As expected, the addition of BDG to cells treated with T-dGuo prevented the metabolism of T-dGuo to 6-thio-guanine-containing ribo-nucleotides, but, unexpectedly, no 6-thio-2'-deoxyguanosine 5'-triphosphate was detected. In cells treated with T-dGuo plus BDG, the major phosphorylated metabolite was T-dGMP. These results indicated that even in the absence of PNP activity, T-dGuo cannot be phosphorylated directly to 6-thio-2'-deoxyguanosine 5'-triphosphate due to the inability of guanylate kinase to utilize T-dGMP as a substrate.
Assuntos
Compostos de Benzil/farmacologia , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tionucleosídeos/toxicidade , Biotransformação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Desoxiguanosina/metabolismo , Desoxiguanosina/toxicidade , Relação Dose-Resposta a Droga , Guanina/farmacologia , Humanos , Tionucleosídeos/metabolismoRESUMO
CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.
Assuntos
Antivirais/metabolismo , Desoxiguanosina/análogos & derivados , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Guanilato Quinases , Células HeLa , Humanos , Núcleosídeo-Fosfato Quinase/metabolismo , Fosforilação , Estereoisomerismo , Especificidade por SubstratoRESUMO
Certain derivatives of 9-deazaguanine that contain arylmethyl, heteroarylmethyl or cycloalkylmethyl groups at the 9-position are potent inhibitors of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). To determine whether these agents can produce metabolically significant inhibition of PNP in cells and in animals, the authors performed pharmacological studies with a representative member of the series, 9-benzyl-9-deazaguanine (BzDAG). BzDAG was a potent inhibitor of PNP from calf spleen (Ki = 12 nM). It was also an effective inhibitor of PNP in cells and in animals as shown by the findings that it 1) inhibited the conversion of inosine to nucleotides in L1210 cells in culture at concentrations that had little effect on the utilization of hypoxanthine; 2) potentiated the toxicity of deoxyguanosine to CCRF-CEM cells in culture; 3) increased the pools of deoxy GTP in CCRF-CEM, Molt-3 and Molt-4 cells that had been treated with deoxyguanosine; 4) prevented the toxicity of 6-thioguanosine to HEp-2 cells in culture; 5) increased the plasma levels of endogenous inosine in rats; and 6) increased the plasma levels of 2',3'-dideoxyinosine in rats that had received BzDAG and dideoxyinosine in combination. Pharmacokinetic analysis of BzDAG in the rat showed it to be 48% orally bioavailable (at a dose of 5 mg/kg). About 95% of BzDAG was protein bound. After i.v. administration of BzDAG (5 mg/kg), more than 50% of the erythrocyte PNP was inhibited for 40 min. These results indicate that the 9-substituted-9-deazaguanines are potent orally active PNP inhibitors and are therefore of potential clinical interest as immunosuppressive and anti-inflammatory agents.
Assuntos
Compostos de Benzil/farmacologia , Guanina/análogos & derivados , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Animais , Compostos de Benzil/metabolismo , Proteínas Sanguíneas/metabolismo , Desoxiguanosina/metabolismo , Desoxiguanosina/farmacologia , Eritrócitos/enzimologia , Guanina/metabolismo , Guanina/farmacologia , Guanosina/análogos & derivados , Guanosina/farmacologia , Inosina/metabolismo , Leucemia L1210/metabolismo , Masculino , Ligação Proteica , Ratos , Ratos Endogâmicos Lew , Tionucleosídeos/farmacologiaRESUMO
The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.
Assuntos
DNA Viral/metabolismo , Desoxiguanosina/análogos & derivados , Herpes Simples/metabolismo , Simplexvirus/metabolismo , Sequência de Bases , DNA Viral/efeitos dos fármacos , Nucleotídeos de Desoxiguanina/metabolismo , Desoxiguanosina/metabolismo , Humanos , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Trítio , Proteínas Virais/antagonistas & inibidoresRESUMO
The carbocyclic analog of 2'-deoxyguanosine [(+-)-2-amino-1,9-dihydro-9-[(1 alpha,3 beta,4 alpha)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-6H-purine-6-one] (2'-CDG) is highly active in cell culture against strains S148 and E377 of herpes simplex virus type 1 (HSV-1), both of which code for thymidine kinase, and much less active against strain BW10168 which is deficient in this enzyme activity. Antiviral activity is associated primarily with the D-enantiomer; the L-enantiomer has much lower but significant activity. The metabolism of racemic 2'-CDG and its D- and L-enantiomers was studied in uninfected HEp-2 cells and in HEp-2 cells infected with the S148 or BW10168 strains of HSV-1. Nucleotides were separated by HPLC, and their elution was monitored by spectrophotometry. The chromatograms of extracts of cells infected with the S148 strain and treated with (+/-)-2'-CDG or D-2'-CDG included a new peak which appeared in the triphosphate region. This peak, the area of which exceeded that of the GTP peak, was shown to be due to the triphosphate of 2'-CDG. The new peak was not observed by HPLC of extracts of uninfected cells treated with (+/-)-2'-CDG or either of its enantiomers, cells infected with the S148 strain and treated with L-2'-CDG, or cells infected with the BW10168 strain and treated with (+/-)-2'-CDG or either of its enantiomers. The results were similar when these studies were performed with uninfected Vero cells and with Vero cells infected with strain S148 of HSV-1. In experiments with D-[8-3H]-2'-CDG, small amounts of phosphates of 2'-CDG could also be detected in uninfected HEp-2 cells and in cells infected with the BW10168 strain of HSV-1. Thus, 2'-CDG apparently is a good substrate for the virus-coded kinase and a very poor substrate for cellular phosphorylating enzymes. The selective phosphorylation of 2'-CDG by the virus-specific kinase presumably is critical for its antiviral activity as it is for that of acyclovir and other acyclic derivatives of guanine.
Assuntos
Antivirais/metabolismo , Desoxiguanosina/análogos & derivados , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Desoxiguanosina/metabolismo , Herpes Simples/metabolismo , Mutação , Nucleotídeos/isolamento & purificação , Fosforilação , Simplexvirus/genética , Timidina Quinase/genéticaRESUMO
3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
AMP Desaminase/fisiologia , Adenina/análogos & derivados , Antibacterianos/farmacologia , Nucleotídeo Desaminases/fisiologia , Nucleotídeos/análise , Tubercidina/análogos & derivados , Tubercidina/farmacologia , Adenina/metabolismo , Adenina/farmacologia , Adenosina Quinase/fisiologia , Alanina/análogos & derivados , Alanina/farmacologia , Aminoglicosídeos , Animais , Sobrevivência Celular/efeitos dos fármacos , Coformicina/farmacologia , Hipoxantina , Hipoxantinas/metabolismo , Camundongos , Fatores de Tempo , Tubercidina/metabolismoRESUMO
The synthesis and characterization of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine (3a) are presented. This compound is a substrate for adenosine deaminase and adenosine kinase. In L1210 cells 3a is converted to 8-aminoinosine monophosphate (4b), apparently by the action of AMP deaminase on the monophosphate of 3a, as well as to the triphosphate derivative of 3a. Pentostatin was used to inhibit adenosine deaminase, and coformycin was used to inhibit AMP deaminase in experiments designed to delineate the metabolic fate of 3a. Pentostatin was without influence on the cytotoxicity of 3a, but coformycin potentiated the cytotoxicity. The potentiation was associated with an increased cellular concentration of phosphates of 3a and a decreased concentration of 4b.
Assuntos
Antineoplásicos/síntese química , Nucleosídeos de Purina/síntese química , AMP Desaminase/antagonistas & inibidores , Inibidores de Adenosina Desaminase , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coformicina/análogos & derivados , Coformicina/farmacologia , Inosina Monofosfato/metabolismo , Cinética , Leucemia L1210/metabolismo , Pentostatina , Nucleosídeos de Purina/farmacologiaRESUMO
Neplanocin A and aristeromycin are carbocyclic adenosine analogs that differ only in that neplanocin A contains a double bond in the carbocyclic ring, whereas this ring in aristeromycin is saturated. We have compared the metabolism and some of the metabolic effects of neplanocin A and synthetic (+/-)-aristeromycin (C-Ado) in murine leukemia L1210 cells in culture. C-Ado, as shown earlier, was not only converted to its own phosphates but also was metabolized to phosphates of carbocyclic guanosine. Both rapidly proliferating and slowly proliferating or resting cells phosphorylated C-Ado, but C-Ado was not converted to phosphates of carbocyclic guanosine in detectable amounts in cells whose growth had reached a plateau. When the metabolism of neplanocin and C-Ado was examined in the same experiment, both analogs were converted to the triphosphate analogs of ATP; no conversion of neplanocin A to the corresponding carbocyclic analogs of guanine nucleotides was detected, whereas C-Ado was converted to the carbocyclic analog of GTP in amounts that approximated the GTP pool. This difference in metabolism was associated with a marked difference in effects of the two analogs on the utilization of hypoxanthine and guanine which was inhibited by C-Ado but not by neplanocin. The failure of neplanocin A to be converted to analogs of guanine nucleotides apparently is the result of poor capacity of its monophosphate to serve as a substrate for AMP deaminase; the Vmax for deamination of neplanocin-5'-monophosphate by this enzyme was only 5% of that for C-Ado monophosphate. In contrast, neplanocin A was a better substrate than C-Ado for adenosine deaminase.