RESUMEN
The nucleoside antibiotic tubercidin displays strong activity against different target organisms, but it is notoriously toxic to mammalian cells. The effects of tubercidin against T. brucei parasites inspired us to synthesize several C7 substituted analogs for in vitro evaluation in order to find suitable hit compounds. C7 Deazaadenosines substituted with electron-poor phenyl groups were found to have micromolar activity against T. brucei in vitro. Replacement of the phenyl for a pyridine ring gave compound 13, with submicromolar potency and much-attenuated cytotoxicity compared to tubercidin. The veterinary pathogen T. congolense was equally affected by 13in vitro. Transporter studies in T. b. brucei indicated that 13 is taken up efficiently by both the P1 and P2 adenosine transporters, making the occurrence of transporter-related resistance and cross-resistance with diamidine drugs such as diminazene aceturate and pentamidine as well as with melaminophenyl arsenicals unlikely. Evaluation of the in vitro metabolic stability of analog 13 indicated that this analog was significantly metabolized in mouse microsomal fractions, precluding further in vivo evaluation in mouse models of HAT.
Asunto(s)
Kinetoplastida/efectos de los fármacos , Tubercidina/química , Animales , Transporte Biológico , Ratones , Proteínas de Transporte de Nucleósidos/metabolismo , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Tubercidina/farmacología , Tubercidina/toxicidadRESUMEN
BACKGROUND: Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms. RESULTS: We show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins. CONCLUSIONS: These AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.
Asunto(s)
Adenosina Quinasa/metabolismo , Adenosina Quinasa/química , Adenosina Quinasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Cricetulus , Exones , Formicinas/toxicidad , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Ratas , Tubercidina/toxicidadRESUMEN
Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB-NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB-NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.
Asunto(s)
Antiparasitarios/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Tioinosina/análogos & derivados , Tionucleótidos/uso terapéutico , Tubercidina/uso terapéutico , Animales , Antiparasitarios/farmacología , Antiparasitarios/toxicidad , Células Cultivadas , Quimioterapia Combinada , Inhibidores Enzimáticos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Schistosoma mansoni/efectos de los fármacos , Tioinosina/farmacología , Tioinosina/uso terapéutico , Tionucleótidos/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Tubercidina/farmacología , Tubercidina/toxicidadRESUMEN
TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain, which was recognized by TOR in this internalization pathway, was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature-sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking.
Asunto(s)
Adenosina/metabolismo , Resistencia a Medicamentos/fisiología , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Adenosina/análogos & derivados , Animales , Línea Celular , Endocitosis/fisiología , Epítopos/fisiología , Humanos , Leishmania mexicana/genética , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Mutación/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Saccharomyces cerevisiae , Tubercidina/metabolismo , Tubercidina/toxicidad , Ubiquitina/metabolismoRESUMEN
Ebola Zaire virus causes lethal hemorrhagic fever in humans, for which there is no effective treatment. A variety of adenosine analogues inhibit the replication of Ebola virus in vitro, probably by blocking the cellular enzyme, S-adenosyl-L-homocysteine hydrolase, thereby indirectly limiting methylation of the 5' cap of viral messenger RNA. We previously observed that adult, immunocompetent mice treated thrice daily for 9 days with 2.2-20 mg/kg of an adenosine analogue, carbocyclic 3-deazaadenosine, were protected against lethal Ebola virus challenge. We now report that a single inoculation of 80 mg/kg or less of the same substance, or of 1 mg/kg or less of another analogue, 3-deazaneplanocin A, provides equal or better protection, without causing acute toxicity. One dose of drug given on the first or second day after virus infection reduced peak viremia more than 1000-fold, compared with mock-treated controls, and resulted in survival of most or all animals. Therapy was less effective when administered on the day of challenge, or on the third day postinfection. Single or multiple doses of the same medications suppressed Ebola replication in severe combined immunodeficient mice, but even daily treatment for 15 consecutive days did not eliminate the infection.
Asunto(s)
Adenosina/análogos & derivados , Antivirales/uso terapéutico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Hidrolasas/antagonistas & inhibidores , S-Adenosilhomocisteína/metabolismo , Tubercidina/análogos & derivados , Adenosina/administración & dosificación , Adenosina/uso terapéutico , Adenosina/toxicidad , Adenosilhomocisteinasa , Animales , Antivirales/administración & dosificación , Antivirales/toxicidad , Línea Celular , Relación Dosis-Respuesta a Droga , Ebolavirus/efectos de los fármacos , Ebolavirus/fisiología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/toxicidad , Femenino , Hidrolasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Tubercidina/administración & dosificación , Tubercidina/uso terapéutico , Tubercidina/toxicidadRESUMEN
We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.
Asunto(s)
Antivirales/farmacología , Mengovirus/efectos de los fármacos , Tubercidina/análogos & derivados , Replicación Viral/efectos de los fármacos , Adenosina/análogos & derivados , Animales , Embrión de Pollo , Células HeLa , Humanos , Mengovirus/genética , Mengovirus/fisiología , Virus de la Enfermedad de Newcastle/efectos de los fármacos , Nucleósidos/farmacología , Virus Sindbis/efectos de los fármacos , Sobreinfección , Tubercidina/metabolismo , Tubercidina/farmacología , Tubercidina/toxicidad , Virus Vaccinia/efectos de los fármacosRESUMEN
Employing thymidylate synthase (TS) (5, 10-CH2-H4PteGlu: dUMP C-methyltransferase, EC 2.1.1.45), a key target enzyme in chemotherapy, the biological activity of the beta-carboline-benzoquinolizidine alkaloid deoxytubulosine (DTB) isolated from the Indian medicinal plant Alangium lamarckii has been evaluated and assessed for the first time. The TS employed in the present studies was purified from Lactobacillus leichmannii. The DTB was demonstrated to exhibit potent cytotoxicity and inhibited the cell growth of L. leichmannii, and DTB potently inhibited TS activity (IC50 = 40 microM). The DTB concentrations > 80 microM resulted in a total loss of the TS activity, thus suggesting that the beta-carboline-benzoquinolizidine alkaloid is a promising potential antitumor agent. The DTB binding to TS appears to be irreversible and tight through a possible covalent linkage. Although DTB strongly binds to DNA, it is not known whether DTB binds to RNA associated with TS. Inhibition kinetics showed that TS has a Ki value of 7 x 10(-6) M for DTB and that the inhibition is a simple linear "noncompetitive" type.
Asunto(s)
Emetina/análogos & derivados , Emetina/farmacología , Inhibidores de Crecimiento/farmacología , Lactobacillus/enzimología , Plantas Medicinales/química , Quinolizinas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/metabolismo , Tubercidina/farmacología , División Celular/efectos de los fármacos , Emetina/toxicidad , Cinética , Lactobacillus/efectos de los fármacos , Lactobacillus/crecimiento & desarrollo , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Quinolizinas/toxicidad , Tubercidina/toxicidadRESUMEN
Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [3H]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 +/- 0.9 x 10(5)/cell, Kd = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [3H]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500,000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak M(r) value, 63,000) than did those of human erythrocytes (peak M(r) value, 53,000). This difference in apparent M(r) was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like mRNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a protein identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of 3H-labeled adenosine, uridine and thymidine by 50% (IC50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which accounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (IC50 > 1.0 microM) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37 degrees C) for the es- and ei-mediated processes differed markedly. Values for transport of uridine by the es- and ei-mediated processes were, respectively: K(m) = 229 +/- 39 and 1077 +/- 220 microM; Vmax, 186 +/- 31 and 40 +/- 5 pmol/microliter cell water/sec. Values for transport of adenosine by the es and ei-mediated processes were, respectively, 61 +/- 9 and 133 +/- 17 microM; Vmax, 70 +/- 5 and 23 +/- 8 pmol/microlitere cell water/sec. The ei-mediated process, although small, was of pharmacologic importance since K562 cells could not be protected by NBMPR (10 microM) from the cytotoxic effects of tubercidin (7-deazaadenosine).
Asunto(s)
Proteínas Portadoras/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Proteínas de la Membrana/metabolismo , Tioinosina/análogos & derivados , Adenosina/metabolismo , Marcadores de Afinidad , Sitios de Unión , Northern Blotting , División Celular , Línea Celular , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Eritrocitos/metabolismo , Amplificación de Genes , Humanos , Técnicas In Vitro , Proteínas de Transporte de Nucleósidos , Reacción en Cadena de la Polimerasa , Unión Proteica/efectos de los fármacos , Tioinosina/metabolismo , Tioinosina/farmacología , Timidina/metabolismo , Tubercidina/toxicidad , Uridina/metabolismoRESUMEN
3-Deazaadenosine (DZA), 3-deaza-(+/-)-aristeromycin (DZAri), and 3-deazaneplanocin A (DZNep) are powerful modulators of cellular processes. When tested against H9 cells infected acutely with two different strains of human immunodeficiency virus 1 (HIV-1) and in the chronically infected monocytoid cell lines U1 and THP-1, the 3-deazanucleosides caused a marked reduction in p24 antigen production. Similar reductions in p24 antigen were seen in phytohemagglutinin-stimulated peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Strikingly, in comparing the therapeutic indices between the paired pre- and post-3'-azido-3'-deoxythymidine (AZT) treatment HIV-1 isolates, DZNep and neplanocin A showed an increase of 3- to 18-fold in their potency against AZT-resistant HIV-1 isolates. In H9 cells treated with DZNep and DZAri, the formation of triphosphate nucleotides of DZNep and DZAri was observed. The mode of action of DZNep and DZAri appears complex, at least in part, at the level of infectivity as shown by decreases in syncytia formation in HIV-1-infected H9 cells and at the level of transcription as both drugs inhibited the expression of basal or tat-induced HIV-1 long terminal repeat chloramphenicol acetyltransferase activity in stably transfected cell lines. Since DZNep induced in H9 cells a rapid expression of nuclear binding factors that recognize the AP-1 transcription site, the anti-HIV-1 activity of the DZA analogs could partly be the induction of critical factors in the host cells. Thus, the 3-deazanucleoside drugs belong to an unusual class of anti-HIV-1 drugs, which may have therapeutic potential, in particular against AZT-resistant strains.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Tubercidina/farmacología , Zidovudina/farmacología , Síndrome de Inmunodeficiencia Adquirida/sangre , Antivirales/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Farmacorresistencia Microbiana , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/biosíntesis , Seronegatividad para VIH , VIH-1/fisiología , Humanos , Monocitos/efectos de los fármacos , Monocitos/patología , Monocitos/virología , Estereoisomerismo , Relación Estructura-Actividad , Factores de Transcripción/análisis , Factores de Transcripción/biosíntesis , Tubercidina/toxicidadRESUMEN
Cell death initiated by the adenosine analog 3-deazaadenosine (c3 Ado) was studied in human promyelocytic leukemia HL-60 cells. A rapid decrease in cell number was seen after 4-hr exposure to 50-100 microM c3 Ado. The dominating mode of cell death was apoptosis as demonstrated by condensation and fragmentation of the nucleus, formation of apoptotic bodies and endonucleolytic degradation of DNA. Four hour treatment with 100 microM c3 Ado resulted in a reduction of early S-phase cells, and appearance of cells with a lower DNA and protein content than that of the G1 population. Whereas 25 and 50 microM c3 Ado only initiated apoptosis in S-phase cells, 75 and 100 microM c3 Ado also initiated apoptosis in G1- and G2 + M-phase cells, suggesting different mechanisms for cell death at different concentrations. Apoptosis initiated by 100 microM c3 Ado was completely inhibited by 1 mM ZnCl2. Addition of homocysteine thiolactone (Hcy) partly inhibited cell death by c3 Ado. Light microscopic examination of cultures treated with 100 microM c3 Ado and 1 mM Hcy showed nuclear condensation and fragmentation consistent with the first stage in apoptosis, however, only a minor formation of apoptotic bodies took place in these cultures compared to that observed in cultures treated with 100 microM c3 Ado alone. The modifying action of Hcy on c3 Ado initiated apoptosis in HL-60 cells and this suggests that c3 Ado and 3-deazaadenosylhomocysteine (c3 AdoHcy) interact with different targets during initiation and progression of cell death in this cell line.
Asunto(s)
Homocisteína/farmacología , Tubercidina/toxicidad , Apoptosis/efectos de los fármacos , Muerte Celular , Cloruros/farmacología , Citometría de Flujo , Humanos , Fase S , Tubercidina/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de los fármacos , Compuestos de Zinc/farmacologíaRESUMEN
Effects of the transmethylation inhibitors 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on cell cycle and cyclic AMP (cAMP) concentrations in human promyelocytic leukemia cells (HL-60) were studied by flow cytometry and radioimmunoassay techniques. Previously described cell cycle accumulations, after incubation with drugs (25 microM) for two cell doublings (36 hr), were localized to G1 and G2 after incubation with c3Ado and c3Ari, respectively. cAMP levels were elevated in cells treated with c3Ado (35%) and c3Ari (92%) for 36 hr. Addition of the phosphodiesterase (PDE) inhibitor theophylline, increased cAMP levels further, while cAMP responsiveness to the beta-adrenergic stimulator isoproterenol was attenuated after c3Ado and c3Ari incubation. Homocysteine thiolactone (Hcy) alone reduced cell growth slightly (5%) and increased cAMP levels (17%). Hcy increased the growth inhibitory effects of c3Ado, while no modulating effect was seen in combination with c3Ari, nor did Hcy counteract the effects on the cell cycle perturbations. The results suggest that c3Ado- and c3Ari-induced cell cycle accumulation is, at least in part, mediated through cAMP elevation, possibly due to PDE inhibition secondary to S-adenosyl-homocysteine hydrolase inhibition and S-adenosyl-homocysteine build-up.
Asunto(s)
Adenosina/análogos & derivados , AMP Cíclico/metabolismo , Leucemia/enzimología , Metiltransferasas/antagonistas & inhibidores , Tubercidina/farmacología , Adenosina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Citometría de Flujo , Fase G1 , Fase G2 , Homocisteína/farmacología , Humanos , Tubercidina/toxicidadRESUMEN
In an attempt to elucidate the types of nucleoside transporters present in bone marrow stem cells, this study examined the effect of nucleoside transport inhibitors on the toxicity of nucleoside analogs and on the salvage of thymidine by mouse bone marrow granulocyte and macrophage progenitor cells using the CFU-GM assay. Concentrations of NBMPR (nitrobenzylmercaptopurine riboside) as low as 10 nM protected these cells from the toxicity of the adenosine analog tubercidin and provided a partial block of thymidine-rescue of the granulocyte-macrophage progenitor cells from methotrexate toxicity. Dipyridamole had similar effects but generally required higher concentrations. These results suggested that the major nucleoside transporter in these cells is the NBMPR-sensitive equilibrative carrier, es. In contrast to the results with tubercidin, the toxicity of 2-chlorodeoxyadenosine was increased 8- to 10-fold by 1 microM NBMPR. These results suggested that the bone marrow granulocyte-macrophage progenitor cells also have a concentrative nucleoside transporter that is capable of pumping 2-chlorodeoxyadenosine into the cells while efflux of the nucleoside via es is blocked by NBMPR.
Asunto(s)
Antimetabolitos Antineoplásicos/toxicidad , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteínas de Transporte de Nucleósidos/antagonistas & inhibidores , Tioinosina/análogos & derivados , Timidina/metabolismo , Animales , Antineoplásicos/toxicidad , Transporte Biológico , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Cladribina/toxicidad , Femenino , Granulocitos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Macrófagos/metabolismo , Metotrexato/toxicidad , Ratones , Ratones Endogámicos CBA , Tioinosina/farmacología , Tubercidina/toxicidadRESUMEN
The toxicity and antiviral efficacy of carbocyclic 3-deazaadenosine (Cc3Ado) against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) virus infections were tested in tissue culture and in cotton rats. The mean median efficacious dose (ED50) of Cc3Ado in HEp2 cells against RSV and PIV3 was 9 and 14 micrograms/ml, respectively. These values were 85- and 55-fold less than the median inhibitory (toxic) dose (ID50) of Cc3Ado in this cell line (750 micrograms/ml), and similar to values obtained for ribavirin. Cc3Ado exhibited no significant antiviral activity against influenza A, influenza B, adeno type 5 or adeno type 7 viruses (all ED50 were greater than 1000 micrograms/ml). In cotton rats, animals given greater than or equal to 1 mg/kg/day Cc3Ado intraperitoneally on days 1, 2 and 3 after experimental challenge with virus, consistently had significant reductions in pulmonary RSV and PIV3 titers compared to pulmonary virus titers in comparably treated control animals. The minimum efficacious dose of ribavirin given under the same conditions was 30 mg/kg/day. Cc3Ado was also efficacious in cotton rats when given orally by gavage, or when different administration schedules were used. The median efficacious dose of Cc3Ado when given orally was 10 mg/kg/day. No significant toxic effects were noted in cotton rats, even in animals given 20 mg/kg daily for eight consecutive days.
Asunto(s)
Antivirales/toxicidad , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Virus Sincitiales Respiratorios/efectos de los fármacos , Tubercidina/análogos & derivados , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Virus de la Parainfluenza 3 Humana/crecimiento & desarrollo , Infecciones por Paramyxoviridae/tratamiento farmacológico , Infecciones por Paramyxoviridae/patología , Ratas , Virus Sincitiales Respiratorios/crecimiento & desarrollo , Infecciones por Respirovirus/tratamiento farmacológico , Infecciones por Respirovirus/patología , Tubercidina/farmacología , Tubercidina/toxicidadRESUMEN
Host toxicity of the dose regimen of tubercidin (7-deazaadenosine) plus nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) used in combination therapy of schistosomiasis (M. H. el Kouni, D. Diop, and S. Cha, Proc. Natl. Acad. Sci. USA 80:6667-6670, 1983; M. H. el Kouni, N. J. Messier, and S. Cha, Biochem. Pharmacol. 36:3815-3821, 1987) was examined in vivo in mice and in vitro with human bone marrow progenitor cells. Four successive daily intraperitoneal injections of tubercidin at 5 mg/kg per day produced 100% mortality in mice within 3 to 5 days following the first injection, with massive peritonitis and intestinal obstruction secondary to abdominal adhesions. Coadministration of NBMPR-P (25 mg/kg per day) protected the mice from the lethality of tubercidin and allowed the repetition of the regimen for a second time with 100% survival until the mice were sacrificed 22 days following the first injection. Blood chemistry, hematological studies, and histological examinations showed no evidence for injury to the liver, kidney, spleen, pancreas, mesentery, or peritoneal mesothelium. In vitro, tubercidin alone had a direct dose-dependent inhibitory effect on myeloid and erythroid human bone marrow progenitor cells, and consistent inhibition (50%) of granulocyte-macrophage CFU (CFU-GM) and erythroid burst-forming units (BFU-E) occurred at 2 to 3 nM tubercidin. At higher doses, BFU-E were more sensitive to tubercidin toxicity than CFU-GM. Complete inhibition (99%) of BFU-E colonies occurred at 10 nM tubercidin, while complete inhibition of CFU-GM occurred at 100 nM. NBMPR-P at 10 to 100 nM protected CFU-GM and BFU-E from tubercidin toxicity in a dose-dependent matter.
Asunto(s)
Antibacterianos/toxicidad , Inosina/análogos & derivados , Esquistosomiasis/tratamiento farmacológico , Tioinosina/análogos & derivados , Tionucleótidos/farmacología , Tubercidina/toxicidad , Aminoglicósidos , Animales , Ensayo de Unidades Formadoras de Colonias , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Técnicas In Vitro , Ratones , Esquistosomiasis/patología , Tioinosina/farmacología , Tioinosina/uso terapéutico , Tionucleótidos/uso terapéutico , Tubercidina/antagonistas & inhibidores , Tubercidina/uso terapéuticoRESUMEN
In contrast to their effects on mammalian cells, the nucleoside transport inhibitors nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) dilazep, benzylacyclouridine (BAU), and to a lesser extent, dipyridamole have no significant effect on the in vitro uptake of adenosine analogues by Schistosoma mansoni [el Kouni and Cha, Biochem. Pharmac. 36, 1099 (1987)]. Coadministration of either NMBPR-P or dilazep with potentially lethal doses of tubercidin (7-deazaadenosine), nebularine or 9-deazaadenosine protected mice from the toxicity of these adenosine analogues. Dipyridamole caused partial protection, whereas BAU did not protect the animals from this toxicity. Toyocamycin caused delayed mortality (after 16 weeks) which could not be prevented by coadministration of NBMPR-P. In S. mansoni infected mice, treated with the combination of NBMPR-P and 9-deazaadenosine was not effective against the parasite. On the other hand, the combinations of NBMPR-P or dilazep with either tubercidin or nebularine were highly toxic to the parasite but not the host. Combination therapy caused a marked reduction in the number of pairing of worms. Effectiveness of combination therapy could also be noted by a drastic decrease in the number of eggs in the liver and small intestine. All eggs found were dead, indicating a direct effect on ovigenesis. Although dipyridamole was less effective than NBMPR-P or dilazep in protecting the host from the toxicity of tubercidin or nebularine, the combinations with dipyridamole produced similar significant therapeutic effects in animals that survived. Mice receiving the combination of tubercidin (or nebularine) plus NBMPR-P or dilazep, as well as those that survived the combination with dipyridamole, appeared healthy and were found to have normal size livers and spleens. These results suggest that highly selective toxicity against schistosomes can be achieved by coadministration of various nucleoside transport inhibitors with adenosine analogues.
Asunto(s)
Nucleósidos de Purina/uso terapéutico , Esquistosomiasis/tratamiento farmacológico , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Dilazep/uso terapéutico , Dipiridamol/uso terapéutico , Quimioterapia Combinada , Femenino , Ratones , Nucleósidos de Purina/toxicidad , Ribonucleósidos/toxicidad , Schistosoma mansoni/metabolismo , Tioinosina/análogos & derivados , Tioinosina/uso terapéutico , Tionucleótidos/uso terapéutico , Toyocamicina/toxicidad , Tubercidina/toxicidad , Uracilo/análogos & derivados , Uracilo/uso terapéuticoRESUMEN
Coadministration of nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) with high doses of tubercidin by i.p. injection into Schistosoma japonicum infected mice beginning 5 weeks post-infection was highly toxic to the parasite but not the hose. Combination therapy resulted in a striking reduction in the number of worms, and the few worms that could be found were stunted. Combination therapy also caused a drastic reduction in the number of eggs in the livers (from 86,500 to 2,800 eggs/liver) and intestines (from 2,200 to 74 eggs/cm2), and 95% of eggs that were found were dead, indicating the termination of oviposition. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal size livers and spleens. These results demonstrate that by combining NBMPR-P with tubercidin high selective toxicity against S. japonicum can be achieved, as was shown previously with S. mansoni.
Asunto(s)
Inosina/análogos & derivados , Ribonucleósidos/administración & dosificación , Esquistosomiasis Japónica/tratamiento farmacológico , Tioinosina/análogos & derivados , Tionucleótidos/administración & dosificación , Tubercidina/administración & dosificación , Animales , Quimioterapia Combinada , Femenino , Riñón/efectos de los fármacos , Ratones , Ratones Endogámicos , Tioinosina/administración & dosificación , Tioinosina/toxicidad , Tionucleótidos/toxicidad , Tubercidina/toxicidadRESUMEN
(+/-)-3-(4-Amino-1H-pyrrolo[2,3-d]pyrimidin-1-yl)-5-(hydroxymethyl)- 1 alpha,2 alpha,3 beta,5 beta)-1,2-cyclopentanediol (9), the carbocyclic analogue of tubercidin, prepared from (+/-)-3-amino-5-(hydroxymethyl)-(1 alpha,2 alpha,3 beta,5 beta)- 1,2-cyclopentanediol (6), is cytotoxic to cells containing adenosine kinase but not to cells that do not, indicating that its activity depends on phosphorylation. Although inactive against P388 leukemia in mice and against herpes and influenza viruses in vitro, it showed marginal activity against respiratory syncytial, vesicular stomatitis, and rhino viruses in vitro.
Asunto(s)
Antineoplásicos/síntesis química , Ribonucleósidos/síntesis química , Tubercidina/síntesis química , Animales , Antivirales/síntesis química , Carcinoma de Células Escamosas , Línea Celular , Humanos , Indicadores y Reactivos , Neoplasias Laríngeas , Leucemia L1210/tratamiento farmacológico , Espectroscopía de Resonancia Magnética , Ratones , Relación Estructura-Actividad , Tubercidina/análogos & derivados , Tubercidina/toxicidadRESUMEN
A simple technique for the isolation of Leishmania donovani and Leishmania tropica promastigote clones derived from a single cell involves the use of semi-solid agar. Both species of Leishmania form discrete colonies at high efficiency in completely defined medium lacking serum. Visible colonies appear between eight and fourteen days. Viability of colonies transferred from semi-solid agar to liquid suspension culture is 100%. Using these techniques, we have isolated clonal populations of cells resistant to tubercidin and formycin b.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Formicinas/toxicidad , Leishmania/genética , Ribonucleósidos/toxicidad , Tubercidina/toxicidad , Agar , Animales , Células Clonales , Medios de Cultivo , Resistencia a Medicamentos , Leishmania/efectos de los fármacos , Leishmania/aislamiento & purificación , Especificidad de la EspecieRESUMEN
The coadministration of tubercidin and ethidium to mice infected with Trypanosoma gambiense gave a better parasite clearance than either of the single drugs. The combination was also more toxic to the mice but the inclusion of nitrobenzylthioinosinate in the therapy significantly alleviated the toxicity of the drug combination. Nitrobenzylthioinosinate per se had no trypanocidal activity and did not affect the trypanocidal action of the drugs. The biochemical basis for the nitrobenzylthioinosinate action appears to be due to the reduction of access of the drugs to tissues or organs sensitive to the toxic drugs. The potential for the use of this compound with nucleoside analogue compounds in the therapy of African trypanosomiasis is suggested.
Asunto(s)
Etidio/farmacología , Inosina/análogos & derivados , Ribonucleósidos/farmacología , Tioinosina/análogos & derivados , Tionucleótidos/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Tripanosomiasis Africana/parasitología , Tubercidina/farmacología , Animales , Sinergismo Farmacológico , Quimioterapia Combinada , Etidio/uso terapéutico , Etidio/toxicidad , Masculino , Ratones , Tioinosina/farmacología , Tioinosina/uso terapéutico , Tionucleótidos/uso terapéutico , Trypanosoma brucei gambiense/crecimiento & desarrollo , Tripanosomiasis Africana/tratamiento farmacológico , Tubercidina/uso terapéutico , Tubercidina/toxicidadRESUMEN
The i.v. administration of tubercidin, an analog of adenosine, in a single dose of 45 mg/kg caused death in about 90% of B10D2F1 mice so treated. Serum and urine analysis, as well as histological examination of tissues, related the lethality of tubercidin to hepatic injury, which was markedly reduced when mice were treated with the inhibitor of nucleoside transport, nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), at i.p. doses higher than 10 mg/kg 30 min prior to tubercidin injection. With high NBMPR-P doses (100 mg/kg, i.p.) followed by tubercidin injection (45 mg/kg, i.v.), kidney damage and high mortality occurred. The tissue distribution of 3H following (( G-3H]tubercidin administration paralleled hepatic or renal injury: NBMPR-P treatment decreased the content of tubercidin-derived 3H in liver and increased that in kidney. Furthermore, the half-life of the decline in tubercidin levels in serum during the first minute after[3H]tubercidin administration was longer in NBMPR-P-treated mice (26 sec) than in untreated mice (10 sec), with the result that 3H levels in serum were more than ten times higher in the former than in the latter at an early stage during the distribution of tubercidin. Within 15 min after i.p. administration, the tissue distribution of (( 3H]tubercidin was complete. The i.p. administration of tubercidin caused ascites and the appearance of amylase in the peritoneal fluid evidently because of peritonitis and pancreatic injury. Administration of NBMPR-P by the i.p. route, but not by the i.v. route, prevented these injuries and shifted the LD50 of i.p. injected tubercidin (5 mg/kg) to markedly higher values (a 4-fold increase with NBMPR-P at 100 mg/kg). The protection of mice by NBMPR-P against lethal injuries caused by i.p. injected tubercidin was consistent with the inhibition by NBMPR-P of tubercidin accumulation in mesentery and pancreas. The tissue specificity of the NBMPR-P influence on the tissue distribution of tubercidin may reflect differences in NBMPR-P pharmacokinetics and/or in properties of the nucleoside permeation mechanism among various tissues.