RESUMO
Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.
Assuntos
Antivirais/metabolismo , Tratamento Farmacológico da COVID-19 , Descoberta de Drogas , SARS-CoV-2/efeitos dos fármacos , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/metabolismo , Adenina/farmacologia , Adenosina/efeitos adversos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Monofosfato de Adenosina/efeitos adversos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/efeitos adversos , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Amidas/efeitos adversos , Amidas/metabolismo , Amidas/farmacologia , Antivirais/efeitos adversos , Antivirais/farmacologia , COVID-19/metabolismo , Cloroquina/efeitos adversos , Cloroquina/análogos & derivados , Cloroquina/metabolismo , Cloroquina/farmacologia , Desenho de Fármacos , Humanos , Redes e Vias Metabólicas , Simulação de Acoplamento Molecular , Nitrocompostos/efeitos adversos , Nitrocompostos/metabolismo , Nitrocompostos/farmacologia , Pirazinas/efeitos adversos , Pirazinas/metabolismo , Pirazinas/farmacologia , Pirrolidinas/efeitos adversos , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ribavirina/efeitos adversos , Ribavirina/metabolismo , Ribavirina/farmacologia , SARS-CoV-2/metabolismo , Tiazóis/efeitos adversos , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Ribavirin is an important component of the treatment for hepatitis C virus (HCV) infection and, in combination with the new direct-acting antiviral (DAA) agents, comprises the major current therapeutic regimens. This study evaluated the cytotoxicity and chromosomal instability induced by ribavirin using the in vitro cytokinesis-block micronucleus cytome (CBMN-Cyt) assay in two cell lines with different expression levels of drug-metabolizing enzymes: human hepatocellular carcinoma cells (HepG2) and Chinese hamster ovary (CHO-K1) cells. HepG2 cells were treated with nine concentrations (from 15.3 µg/ml to 3.9 mg/ml) and CHO-K1 cells were exposed to eight concentrations (from 15.3 µg/ml to 1.9 mg/ml) of ribavirin for 24 h. Ribavirin inhibited cell proliferation in both cell lines, but at different concentrations: 3.9 mg/ml in HepG2 and 244.2 µg/ml in CHO-K1 cells. No significant differences were observed regarding aspects of cell death in HepG2 and CHO-K1 cells, reflecting the absence of cytotoxic effects associated to ribavirin. Ribavirin did not increase the frequency of nucleoplasmic bridges (NPBs) and nuclear bud (NBUD). However, when compared to the negative control, a significant increase in micronuclei (MNi) frequency was observed in both cell lines. However, chromosomal instability was induced by higher concentrations of ribavirin in HepG2 cells (from 61.1 to 976.8 µg/ml), compared with CHO-K1 cells (15.3 and 30.5 µg/ml). These results demonstrate the potential of ribavirin to promote chromosomal instability, and suggest that cells with different expressions of drug-metabolizing enzymes show different susceptibility to ribavirin effects.
Assuntos
Antivirais/toxicidade , Proliferação de Células/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Ribavirina/toxicidade , Animais , Antivirais/metabolismo , Apoptose/efeitos dos fármacos , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Inativação Metabólica , Testes para Micronúcleos , Ribavirina/metabolismoRESUMO
Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.
Assuntos
RNA Polimerases Dirigidas por DNA/análise , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Vírion/enzimologia , Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , RNA Viral/biossíntese , Ribavirina/metabolismo , Ribonucleotídeos/metabolismo , Montagem de VírusRESUMO
Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.
Assuntos
Antivirais/metabolismo , Enzimas Imobilizadas/metabolismo , Proteínas de Escherichia coli/metabolismo , Fosfotransferases/metabolismo , Ribavirina/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Excipientes , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/isolamento & purificação , TemperaturaRESUMO
A infecção pelo vírus da hepatite C (VHC) é comumente assintomática e apresenta uma elevada taxa de cronicidade, podendo evoluir para cirrose e carcinoma hepatocelular. O diagnóstico da hepatite C é realizado através da pesquisa de anticorpos pelo teste de ELISA (Enzyme Linked Immunosorbent Assay) e confirmado por testes suplementares, tais como o RIBA (Recombinant Immunoblot Assay) e westernblot e teste confirmatório, como a pesquisa do VHC-RNA. O objetivo deste trabalho foi avaliar a eficácia do RIBA no diagnóstico da infecção pelo VHC em doadores de sangue com anti-VHC reagente. Foram analisadas 102 amostras com resultado de anti-VHC reagente naEMOBA, utilizando-se o teste anti-VHC Architect Abbott por quimioluminescência para detecção dos anticorpos anti-VHC, o RIBA III (CHIRON) como teste suplementar para as amostras anti-VHC reagentes e indeterminadas e a Reação em Cadeia da Polimerase (RT-PCR) convencional ou em tempo real (Amplicor Roche) para detecção do VHC- NA. As amostras com VHC-RNA detectável foram genotipadas por hibridização reversa (LIPA; SIEMENS). Das 102 amostras analisadas no LACEN, 38,2% (39/102) foram reagentes, 57,8% (59/102) foram não reagentes e 3,9% (4/102) foram indeterminadas para o anti-VHC. Os resultados do RIBA foram 58,1% (25/43) positivos, 9,3% (4/43) negativos e 32,6% (14/43) indeterminados. Todas as amostras com resultado de RIBA indeterminado tiveram carga viral indetectável. As bandas predominantes nas amostras indeterminadas foram c33 e c22. Das amostras indeterminadas no RIBA, repetidas após seis meses com nova coleta, 20% (2/10) negativaram e 71,4% (10/14) permaneceram RIBA indeterminado. Destas, (8/10) continuaram indeterminadas com o mesmo padrão de bandas. O VHC-RNA foi realizado em todas as amostras do estudo (102) e foi detectável em apenas 22,5% (23/102). Todas as amostras com VHC-RNA detectável foram RIBA positivo e tinham índex maior que cinco na relação S/CO. Em apenas duas amostras que tiveram resultado de RIBA positivo, o VHC-RNA não foi detectado. As 23 amostras com VHC-RNA detectável foram genotipadas, sendo 78,3% (18/23) do genótipo 1, 17,4% (4/23) do genótipo 3 e 4,3% (1/23) do genótipo 2. A positividade do anti-VHC associou-se com o uso de droga intranasal (< 0,001), com drogas injetáveis (< 0,001)) e ocorrência de DST (< 0,05). Diante dos resultados encontrados, observa-se que o RIBA apresenta um elevado número de resultados indeterminados, sendo necessária a realização do VHC-RNA para a confirmação da infecção pelo VHC. Indivíduos com resultado de anti-VHC índex menor que cinco e resultado de RIBA indeterminado têm grande probabilidade de não apresentarem o VHC-RNA detectável e portanto, de não estarem infectados pelo VHC, mas devem ser acompanhados sorologicamente, de acordo com o critério médico.
Assuntos
Humanos , Anticorpos Anti-Hepatite C/imunologia , Hepacivirus/patogenicidade , Hepatite C/virologia , RNA , Ribavirina/metabolismoRESUMO
A sensitive and specific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC-MS/MS analysis. (13)C(5)-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245-->113 and 250-->133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01-10 microg/mL with a limit of quantitation of 0.01 microg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 microg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM(ss) cells cultured with RBV.