RESUMO
Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.
Assuntos
Adutos de DNA , Nucleosídeos , Humanos , Ratos , Animais , Nucleosídeos/toxicidade , Cromatografia Líquida , Espectrometria de Massas em Tandem , Dano ao DNA , Fígado , DNA , Organoides , LansoprazolRESUMO
Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues.
Assuntos
Tratamento Farmacológico da COVID-19 , Mutagênicos , Dano ao DNA , Humanos , Mutagênicos/toxicidade , Nucleosídeos/toxicidadeRESUMO
Emerging and re-emerging viruses periodically cause outbreaks and epidemics all over the world, eventually leading to global events such as the current pandemic of the novel SARS-CoV-2 coronavirus infection COVID-19. Therefore, an urgent need for novel antivirals is crystal clear. Here we present the synthesis and evaluation of an antiviral activity of phenoxazine-based nucleoside analogs divided into three groups: (1) 8-alkoxy-substituted, (2) acyclic, and (3) carbocyclic. The antiviral activity was assessed against a structurally and phylogenetically diverse panel of RNA and DNA viruses from 25 species. Four compounds (11a-c, 12c) inhibited 4 DNA/RNA viruses with EC50 ≤ 20 µM. Toxicity of the compounds for the cell lines used for virus cultivation was negligible in most cases. In addition, previously reported and newly synthesized phenoxazine derivatives were evaluated against SARS-CoV-2, and some of them showed promising inhibition of reproduction with EC50 values in low micromolar range, although accompanied by commensurate cytotoxicity.
Assuntos
Antivirais/farmacologia , Vírus de DNA/efeitos dos fármacos , Nucleosídeos/farmacologia , Oxazinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/toxicidade , Linhagem Celular Tumoral , Chlorocebus aethiops , Cães , Humanos , Células Madin Darby de Rim Canino , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/toxicidade , Oxazinas/síntese química , Oxazinas/toxicidade , Relação Estrutura-Atividade , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.
Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos/métodos , Nucleosídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/toxicidade , Linhagem Celular , Chlorocebus aethiops , Coronavirus Humano OC43/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Nucleosídeos/química , Nucleosídeos/toxicidade , Propanolaminas/farmacologia , Sofosbuvir/farmacologia , Células VeroRESUMO
Synthetic nucleoside analogues characterized by a C-C anomeric linkage form a family of promising therapeutics against infectious and malignant diseases. Herein, C-nucleosides comprising structural variations at the sugar and nucleobase moieties were examined for their ability to inhibit both murine and human norovirus RNA-dependent RNA polymerase (RdRp). We have found that the combination of 4-amino-pyrrolo[2,1-f][1,2,4]triazine and its 7-halogenated congeners with either a d-ribose or 2'-C-methyl-d-ribose unit resulted in analogues with good antiviral activity against murine norovirus (MNV), albeit coupled with a significant cytotoxicity. Among this series, 4-aza-7,9-dideazaadenosine notably retained a strong antiviral effect in a human norovirus (HuNoV) replicon assay with an EC50 = 0.015 µM. This study demonstrates that C-nucleosides can be used as viable starting scaffolds for further optimization towards the development of nucleoside-based inhibitors of norovirus replication.
Assuntos
Antivirais/química , Antivirais/farmacologia , Norovirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Pirróis/química , Triazinas/química , Animais , Antivirais/toxicidade , Linhagem Celular , Desenho de Fármacos , Camundongos , Norovirus/fisiologia , Nucleosídeos/toxicidade , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the Saccharomycescerevisiae genomic library of knockout and knockdown (YKO/KD) strains, to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.
Assuntos
Desoxicitidina Quinase/genética , Nucleosídeos/toxicidade , Farmacogenética/métodos , Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina Quinase/metabolismo , Epistasia Genética , Ontologia Genética , Redes Reguladoras de Genes , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fenômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , GencitabinaRESUMO
Acquired Fanconi syndrome has been associated with the long-term ingestion of several nucleoside analogs used to treat chronic hepatitis B virus infection. However, the nucleoside analog entecavir has not been found to cause nephrotoxicity. We report a case of entecavir-induced Fanconi syndrome. Our patient was a 73-year-old man admitted to our hospital because of renal dysfunction. He also presented with hyperaminoaciduria, renal diabetes, phosphaturia, hypophosphatemia, hypokalemia, hypouricemia, and hyperchloremic metabolic acidosis, supporting a diagnosis of Fanconi syndrome. In this case, the cause of Fanconi syndrome was most likely entecavir, which had been administered as needed depending on his renal function for 5 years. After drug discontinuation and replacement with tenofovir alafenamide fumarate therapy once a week, the patient's kidney function recovered and electrolyte anomalies partially improved. We highlight the fact that entecavir may induce severe renal dysfunction, which can cause the development of Fanconi syndrome; therefore, close monitoring of proximal tubular function is recommended during entecavir therapy.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Síndrome de Fanconi/induzido quimicamente , Guanina/análogos & derivados , Hepatite B Crônica/complicações , Nucleosídeos/toxicidade , Acidose/etiologia , Injúria Renal Aguda/sangue , Injúria Renal Aguda/complicações , Injúria Renal Aguda/patologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Idoso , Alanina , Antivirais/uso terapêutico , Síndrome de Fanconi/sangue , Síndrome de Fanconi/tratamento farmacológico , Síndrome de Fanconi/urina , Guanina/efeitos adversos , Guanina/toxicidade , Hepatite B Crônica/tratamento farmacológico , Humanos , Hipopotassemia/etiologia , Hipofosfatemia/etiologia , Masculino , Nucleosídeos/efeitos adversos , Tenofovir/análogos & derivados , Resultado do Tratamento , Suspensão de TratamentoRESUMO
All nucleoside analogues for treating HIV infection, due to their capacity to integrate into and alter human DNA, are experimentally genotoxic to some extent. The long-term oncogenic risk after in utero exposure remains to be determined. Cancer incidence in uninfected children exposed to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) was evaluated, by cross-checking against the National Cancer Registry, in the French perinatal study of children born to HIV+ mothers. Twenty-one cancers were identified in 15,163 children (median age: 9.9 years [interquartile range (IQR): 5.8-14.2]) exposed to at least one NRTI in utero between 1990 and 2014. Five of these children were exposed to zidovudine monotherapy, and 15 to various combinations, seven of which included didanosine. Overall, the total number of cases was not significantly different from that expected for the general population (SIR = 0.8[0.47-1.24]), but the number of cases after didanosine exposure was twice that expected (SIR = 2.5 [1.01-5.19]). Didanosine accounted for only 10% of prescriptions but was associated with one-third of cancers. In multivariate analysis, didanosine exposure was significantly associated with higher risk (HR = 3.0 [0.9-9.8]). This risk was specifically linked to first-trimester exposure (HR = 5.5 [2.1-14.4]). Three cases of pineoblastoma, a very rare cancer, were observed, whereas 0.03 were expected. Two were associated with didanosine exposure. Despite reassuring data overall, there is strong evidence to suggest that didanosine displays transplacental oncogenicity. These findings cannot be extrapolated to other NRTIs, but they highlight the need for comprehensive evaluations of the transplacental genotoxicity of this antiretroviral class. Environ. Mol. Mutagen., 60:404-409, 2019. © 2017 Wiley Periodicals, Inc.
Assuntos
Fármacos Anti-HIV/toxicidade , Exposição Materna , Troca Materno-Fetal/fisiologia , Neoplasias/epidemiologia , Nucleosídeos/toxicidade , Efeitos Tardios da Exposição Pré-Natal/patologia , Adolescente , Fármacos Anti-HIV/uso terapêutico , Criança , Pré-Escolar , Didanosina/uso terapêutico , Didanosina/toxicidade , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Neoplasias/genética , Nucleosídeos/uso terapêutico , Gravidez , Estudos Prospectivos , Inibidores da Transcriptase Reversa/uso terapêutico , Inibidores da Transcriptase Reversa/toxicidade , Risco , Inquéritos e Questionários , Zidovudina/uso terapêutico , Zidovudina/toxicidadeRESUMO
Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.
Assuntos
Antivirais/toxicidade , Nucleosídeos/toxicidade , Testes de Toxicidade , Animais , Antivirais/farmacologia , Humanos , Estrutura Molecular , Nucleosídeos/farmacologia , Vírus/efeitos dos fármacosRESUMO
BACKGROUND: T-705 (Favipiravir) is a broad spectrum antiviral agent approved for stockpiling in Japan and currently in Phase 3 testing in the United States. Against influenza, it acts as a prodrug, converted intracellularly to selectively inhibit viral RNA-dependent RNA polymerase or similar enzymes. This is regarded as a novel antiviral mechanism of action, reducing crossresistance to other existing anti-influenza drugs. OBJECTIVE: To develop new analogs, a class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized in this work. RESULTS: Anti-influenza activity and Anti-HIV activity of these compounds were evaluated. Compound 1a displayed activity against A H1N1 with an IC50 of 40.4 µmol/L. Compound 1b showed weak activity against HIV with a viral suppression rate of 70-80% at 30 µmol/L. CONCLUSION: A class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized, and one of them was identified as a novel scaffold against viral infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Nucleosídeos/farmacologia , Tiofenos/farmacologia , Amidas/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/toxicidade , Cães , Células HEK293 , HIV/efeitos dos fármacos , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Isomerismo , Células Madin Darby de Rim Canino/virologia , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/toxicidade , Pirazinas/farmacologia , Ribavirina/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/toxicidadeRESUMO
4-pyridone-3-carboxamide-1ß-D-ribonucleoside (4PYR) is a derivative of nicotinamide found physiologically in human body fluids that can be metabolized to mono-, di- or triphosphate derivatives (4PYMP, 4PYDP and 4PYTP respectively) and an analogue of NAD - the 1-ß-D-ribonucleoside-4-pyridone-3-carboxamide adenine dinucleotide (4PYRAD) in human cells. The European Uremic Toxin Work Group (EUTox) has classified 4PYR as a uremic toxin that adversely affects endothelium. This study aimed to investigate the metabolism of 4PYR in murine brain microvascular endothelial cells (mBMECs). Incubation of mBMECs with 4PYR was carried out for 0, 24, 48 or 72 h. After incubation, a medium was removed and cellular concentrations of ATP, ADP, NAD, 4PYMP and 4PYRAD were analyzed using reversed-phase HPLC. 4PYR was metabolized by mBMECs to 4PYMP and 4PYRAD that reached concentrations of 2 ± 0.7 and 0.6 ± 0.2 nmol/mg protein (mean ± SEM), respectively, after 72 h incubation. However, unlike with endothelial cells studied so far this process has no effect on energy balance in the cell as indicated by maintained ATP/ADP ratio and adenine and nicotinamide intracellular pools. Further studies are required to explain whether the difference in 4PYR metabolism is related to differences between species or organs.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Microvasos/metabolismo , Nucleosídeos/metabolismo , Piridonas/metabolismo , Adenina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Metabolismo Energético , Humanos , Masculino , NAD/metabolismo , Niacinamida/metabolismo , Nucleosídeos/análise , Nucleosídeos/toxicidade , Piridonas/análise , Piridonas/toxicidadeRESUMO
The widespread use of combined anti-retroviral therapy (cART) has not decreased the prevalence of HIV-1-associated neurocognitive disorder (HAND), a type of neurodegenerative disease, even though cART effectively inhibits virus colonization in the central nervous system. Therefore, anti-retroviral agents cannot be fully excluded from the pathogenesis of HAND. Our previous study reported that long-term nucleoside analogue (NA) exposure induced mitochondrial toxicity in the cortical neurons of HAND patients and mice, but the exact mechanism of NA-associated neurotoxicity has remained unclear. Alteration of autophagy can result in protein aggregation and the accumulation of dysfunctional organelles, which are hallmarks of some neurodegenerative diseases. In this study, we first found increased autophagy in cortical autopsy specimens of AIDS patients. We then found that a low dose of NAs could stimulate autophagy in primary cultured neurons, while a high dose of NAs could induce only neuronal apoptosis. The level of NA-induced Bcl-2 and Bax expressions determined whether neuronal autophagy or apoptosis occurred. Furthermore, the level of NA-induced neuronal apoptosis correlated with the dysfunction of cellular DNA polymerase gamma. Damage-regulated autophagy modulator (DRAM) overexpression was also involved in NA-induced neuronal autophagy. p53 played a role in the regulation of NA-induced neuronal apoptosis, but its role in NA-associated neuronal autophagy was uncertain. Our results suggest that DRAM is involved in the regulation of NA-induced neuronal autophagy in a p53-independent manner. Further research is needed to investigate the underlying mechanism.
Assuntos
Autofagia/fisiologia , Proteínas de Membrana/biossíntese , Neurônios/metabolismo , Nucleosídeos/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Adulto JovemRESUMO
1,3,4-Thiadiazole and sugar-derived molecules have proven to be promising agrochemicals with growth promoting, insecticidal and fungicidal activities. In the research field of agricultural fungicide, applying union of active group we synthesized a new set of 1,3,4-thiadiazole xylofuranose derivatives and all of the compounds were characterized by 1H NMR and HRMS. In precise toxicity measurement, some of compounds exhibited more potent fungicidal activities than the most widely used commercial fungicide Chlorothalonil, promoting further research and development. Based on our experimental data, 3D-QSAR (three-dimensional quantitative structure-activity relationship) was established and investigated using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques, helping to better understand the structural requirements of lead compounds with high fungicidal activity and environmental compatibility.
Assuntos
Fungicidas Industriais/química , Glicosídeos/química , Nucleosídeos/química , Relação Quantitativa Estrutura-Atividade , Tiadiazóis/química , Fungos/classificação , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Fungicidas Industriais/síntese química , Fungicidas Industriais/toxicidade , Glicosídeos/síntese química , Glicosídeos/toxicidade , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/toxicidade , Espectroscopia de Prótons por Ressonância Magnética , Especificidade da Espécie , Eletricidade Estática , Tiadiazóis/síntese química , Tiadiazóis/toxicidadeRESUMO
Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.
Assuntos
Antivirais/toxicidade , Mitocôndrias/efeitos dos fármacos , Ribonucleosídeos/química , Ribonucleosídeos/toxicidade , Adenosina/análogos & derivados , Amidas/toxicidade , Linhagem Celular/efeitos dos fármacos , Citidina/análogos & derivados , Citidina/toxicidade , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/toxicidade , Humanos , Concentração Inibidora 50 , Proteínas Mitocondriais/metabolismo , Nucleosídeos/toxicidade , Pró-Fármacos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Pirazinas/toxicidade , RNA/metabolismo , RNA Mitocondrial , Sofosbuvir/toxicidade , Relação Estrutura-Atividade , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.
Assuntos
Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Nucleosídeos/metabolismo , Pirimidinas/metabolismo , Uridina Quinase/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citidina/metabolismo , Citoplasma/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Nucleosídeos/toxicidade , Fosforilação , Pirimidinas/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Regulação para Cima , Uridina/metabolismo , Uridina Quinase/antagonistas & inibidores , Uridina Quinase/genéticaRESUMO
The bio-reversible protection of nucleoside diphosphates is summarized. The design, hydrolytic characteristics, and the antiviral activity of these prodrugs of NDPs are described. In contrast to earlier attempts, the DiPPro-approach [ß-(bis(acyloxybenzyl) nucleoside diphosphates)] leads to the successful delivery of the desired nucleoside diphosphates. The stability towards hydrolysis is dependent on the specific acyl moieties in the bis(acyloxybenzyl) unit as well as on the particular nucleoside analogue. Hydrolysis studies in aqueous PBS buffer (pH 7.3), 20 % human plasma in PBS, RPMI-1640 culture medium, and CEM cell extracts were carried out. Contrary to a high chemical and plasma stability, the compounds showed a very low half-life in CEM cell extracts, and efficiently released the nucleoside analogues diphosphates, e.g. of AZT, d4T and BVDU. Two additional types of cycloSal- NDP prodrugs were studied but neither proved to be useful as nucleoside diphosphate prodrugs. In summary, the results led to the development of a new series of non-symmetric nucleoside diphosphate prodrugs that selectively delivered the nucleoside diphosphate in cell extracts.
Assuntos
Fármacos Anti-HIV/química , Nucleosídeos/química , Pró-Fármacos/química , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/toxicidade , Proliferação de Células/efeitos dos fármacos , Difosfatos/química , Esterases/metabolismo , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Nucleosídeos/metabolismo , Nucleosídeos/toxicidade , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidadeRESUMO
We recently described a new nicotinamide derivative: 4-pyridone-3-carboxamide ribonucleoside (4PYR) and its conversion to intracellular metabolites (4PYR monophosphate: 4PYMP and 4PYR adenylate diphosphate: 4PYRAD). The aim of this study was to clarify the metabolism and physiological effects of brief exposure to 4PYR in perfused rat heart. Rat hearts were perfused in Langendorff mode. After 15 min equilibration, 100 µM 4PYR (or solvent in controls) was infused into coronary circulation for 5 min. Coronary flow was recorded with electromagnetic flow meter and left ventricular mechanical function was assessed with intraventricular baloon by constructing pressure-volume relations. After perfusion hearts were freeze-clamped and analyzed using HPLC for phosphocreatine, creatine, ATP with metabolites as well as 4PYR metabolites. 4PYR infused into the coronary circulation was rapidly converted in the heart into 4PYMP and 4PYRAD with concentrations reaching 85.6±46.9 and 43.9±6.4 nmol/g dry weight, respectively, while control concentrations were below 20 nmol/g. 4PYR had no effect on baseline coronary flow (11.9±2.3 ml/min versus 11.0±2.7 ml/min in control) or stimulated by shear stress (23.2±4.5 ml/min versus 23.1±5.2 ml/min in control). Both systolic and diastolic left ventricular mechanical function were not affected by 4PYR. No difference was noted for heart rate. Myocardial concentrations of ATP or phosphocreatine were also not affected by 4PYR. We conclude that 4PYR has no immediate effect on coronary endothelium or cardiomyocyte functions such as coronary flow, rhythm, diastolic properties, or contractility despite rapid incorporation into intracellular metabolites. This study also indicates the lack of effect on purinergic receptors.
Assuntos
Vasos Coronários/efeitos dos fármacos , Coração/efeitos dos fármacos , Nucleosídeos/toxicidade , Perfusão , Piridonas/toxicidade , Animais , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/fisiologia , Relação Dose-Resposta a Droga , Coração/fisiologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Nucleosídeos/metabolismo , Piridonas/metabolismo , Ratos , Ratos WistarRESUMO
A series of purine 5'-deoxyphosphonate analogues were designed and synthesized to mimic naturally occurring purine monophosphate from 1,3-dihydroxyacetone as starting material. The discovery of threosyl phosphonate nucleoside (PMDTA, EC50 = 2.53 µM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 2',3'-modified 5'-deoxyversions of the threosyl phosphonate nucleosides. The synthesized 2'-fluoro-3'-hydroxymethyl 5'-deoxythreosyl phosphonic acid nucleoside analogues 14, 18, 23, and 27 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 18 exhibits weak in vitro anti-HIV-1 activity (EC50 = 19.2 µM).
Assuntos
Antivirais/síntese química , Nucleosídeos/síntese química , Ácidos Fosforosos/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/toxicidade , Antivirais/farmacologia , Antivirais/toxicidade , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos/farmacologia , Nucleosídeos/toxicidadeAssuntos
Nucleosídeos/toxicidade , Animais , Capecitabina , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Desoxicitidina/toxicidade , Fluoruracila/análogos & derivados , Fluoruracila/metabolismo , Fluoruracila/toxicidade , Humanos , Nucleosídeos/metabolismo , GencitabinaRESUMO
Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.