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2.
Nucleic Acids Res ; 48(18): 10383-10396, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941607

RESUMO

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.


Assuntos
Bacteriófagos/genética , DNA/genética , Motivos de Nucleotídeos/genética , Pirimidinonas/farmacologia , Pirróis/farmacologia , Bacteriófagos/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , DNA/efeitos dos fármacos , Enzimas de Restrição-Modificação do DNA/efeitos dos fármacos , Escherichia coli/virologia , Edição de Genes , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Sequenciamento por Nanoporos , Motivos de Nucleotídeos/efeitos dos fármacos , Siphoviridae/genética
3.
Biomed Pharmacother ; 131: 110643, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32846329

RESUMO

Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) has been widely spread in the world with a high mortality. Cytokine storm syndrome (CSS) and acute lung injury caused by SARS-CoV-2 infection severely threaten the patients. With the purpose to find effective and low-toxic drugs to mitigate CSS, entecavir and imipenem were identified to reduce TNF-α using a LPS-induced macrophage model from the anti-infective drug library. Entecavir and imipenem efficiently suppressed the release of inflammatory cytokines by partly intervention of NF-κB activity. The acute lung injury was also alleviated and the survival time was prolonged in mice. In addition, entecavir and imipenem inhibited the release of TNF-α and IL-10 in human peripheral blood mononuclear cells (hPBMCs). Collectively, we proposed that entecavir and imipenem might be candidates for the treatment of CSS.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Guanina/análogos & derivados , Imipenem/farmacologia , Pneumonia Viral/tratamento farmacológico , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/virologia , Animais , Infecções por Coronavirus/complicações , Síndrome da Liberação de Citocina/virologia , Citocinas/imunologia , Reposicionamento de Medicamentos , Guanina/farmacologia , Humanos , Interleucina-10/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pandemias , Pneumonia Viral/complicações , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
4.
Mem Inst Oswaldo Cruz ; 115: e190469, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32638832

RESUMO

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.


Assuntos
Antimônio/toxicidade , Proteínas de Escherichia coli/genética , Escherichia coli , Guanina/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Pirofosfatases , Superóxido Dismutase/metabolismo , Animais , Antiprotozoários/farmacologia , Proteínas de Escherichia coli/metabolismo , Guanina/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Coelhos , Ratos , Superóxido Dismutase/genética
5.
J Pharmacol Sci ; 144(1): 43-51, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32653340

RESUMO

Platelet activation is the primary cause of thrombosis. The P2X7 receptor (P2X7R) is a therapeutic target of thrombosis. However, it is still unknown whether P2X7R activation affects platelet thrombus. Our molecular docking results showed that entecavir as a P2X7R antagonist interacted perfectly with the human P2X7R (hP2X7R) in silico simulation studies. Furthermore, our experimental data revealed that entecavir could act as a P2X7R antagonist to exert cytoprotective effects against platelet activation via protecting mitochondrial function, improving lipid peroxidation and increasing antioxidant activity. Correlated with this, entecavir inhibited platelet aggregation, dense-granule secretion, P-selectin expression, integrin activation and Ca2+ increase. In experimental mouse model, entecavir could significantly inhibit arteriovenous thrombosis and prolong the bleeding time. Furthermore, we found that entecavir had no significant effect on prothrombin time (PT), activated partial thrombin time (APTT), thrombin time (TT), fibrinogen (FIB), mean platelet volume (MPV) and platelet counts (PLT). This study demonstrates that entecavir markedly prevents platelet activation and thrombosis through inhibiting P2X7R without affecting coagulation system. Therefore, entecavir may be a potential candidate for treating thrombosis disease.


Assuntos
Guanina/análogos & derivados , Ativação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Receptores Purinérgicos P2X7 , Trombose/prevenção & controle , Animais , Antioxidantes , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Modelos Animais de Doenças , Guanina/farmacologia , Guanina/uso terapêutico , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Agregação Plaquetária/efeitos dos fármacos , Trombose/sangue
6.
Antiviral Res ; 180: 104857, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32562705

RESUMO

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/virologia , Nucleotídeos/farmacologia , Pneumonia Viral/virologia , RNA Replicase/antagonistas & inibidores , Vírus da SARS/enzimologia , Síndrome Respiratória Aguda Grave/virologia , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Betacoronavirus/genética , Cidofovir/química , Cidofovir/farmacologia , Cidofovir/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Didesoxinucleosídeos/química , Didesoxinucleosídeos/farmacologia , Didesoxinucleosídeos/uso terapêutico , Ganciclovir/química , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Guanina/análogos & derivados , Guanina/química , Guanina/farmacologia , Guanina/uso terapêutico , Nucleotídeos/química , Nucleotídeos/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Estavudina/química , Estavudina/farmacologia , Estavudina/uso terapêutico , Valganciclovir/química , Valganciclovir/farmacologia , Valganciclovir/uso terapêutico
7.
Arch Med Res ; 51(3): 233-244, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32139108

RESUMO

OBJECTIVE: To evaluate the anti-cancer effect of unmethylated cytosine-phosphorothioate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) on human bladder cancer UM-UC-3 cells, our study was carried out. METHODS: The viability of cells (UM-UC-3, T24 and SV-HUC-1) with CpG ODN treatments was examined by cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot. RESULTS: Experimental results showed that the viability of human bladder cancer cells (UM-UC-3 and T24) with CpG ODN treatment was decreased and the viability of human normal urothelial cells (SV-HUC-1) with CpG ODN treatment was increased with time-dependance manner. Moreover, CpG ODN increased the apoptosis rate of UM-UC-3 cells and arrested more cells in G0G1 phase. Furthermore, the expression of caspase-3, p53 and Bax were increased and the expression of Bcl-2 was decreased with CpG ODN treatment on UM-UC-3 cells. CONCLUSION: CpG ODN promoted the proliferation of normal urinary transitional epithelial cells (SV-HUC-1) and inhibited the cell viability of human bladder cancer cells (UM-UC-3 and T24) in vitro. CpG ODN induced the apoptosis of human bladder cancer (UM-UC-3) cells in a cascade progress via enhancing the expression of caspase-3, p53 and Bax, and inhibiting the expression of Bcl-2 with significant time-dependancy. CpG ODN inhibited cell cycle distribution of human bladder cancer (UM-UC-3) cells with more cells were arrested in G0G1 phase. This study suggested that the CpG ODN is the potential candidate on human bladder cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citosina/farmacologia , Células Epiteliais/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Guanina/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteína X Associada a bcl-2/metabolismo
8.
Nat Commun ; 11(1): 714, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024835

RESUMO

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Riboswitch/genética , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico , DNA Complementar , Regulação da Expressão Gênica/efeitos dos fármacos , Guanina/farmacologia , Células HEK293 , Vírus Delta da Hepatite/genética , Humanos , RNA Catalítico/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Riboswitch/efeitos dos fármacos , Biologia Sintética/métodos , Tetraciclina/farmacologia
9.
Sci Rep ; 10(1): 3021, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080249

RESUMO

Chronic hepatitis B virus (HBV) infection is a major public health problem that affects millions of people worldwide. Nucleoside analogue reverse transcriptase (RT) inhibitors, such as entecavir (ETV) and lamivudine (3TC), serve as crucial anti-HBV drugs. However, structural studies of HBV RT have been hampered due to its unexpectedly poor solubility. Here, we show that human immunodeficiency virus type-1 (HIV-1) with HBV-associated amino acid substitutions Y115F/F116Y/Q151M in its RT (HIVY115F/F116Y/Q151M) is highly susceptible to ETV and 3TC. Additionally, we experimentally simulated previously reported ETV/3TC resistance for HBV using HIVY115F/F116Y/Q151M with F160M/M184V (L180M/M204V in HBV RT) substituted. We determined crystal structures for HIV-1 RTY115F/F116Y/Q151M:DNA complexed with 3TC-triphosphate (3TC-TP)/ETV-triphosphate (ETV-TP)/dCTP/dGTP. These structures revealed an atypically tight binding conformation of 3TC-TP, where the Met184 side-chain is pushed away by the oxathiolane of 3TC-TP and exocyclic methylene of ETV-TP. Structural analysis of RTY115F/F116Y/Q151M/F160M/M184V:DNA:3TC-TP also demonstrated that the loosely bound 3TC-TP is misaligned at the active site to prevent a steric clash with the side chain γ-methyl of Val184. These findings shed light on the common structural mechanism of HBV and HIV-1 resistance to 3TC and ETV and should aid in the design of new agents to overcome drug resistance to 3TC and ETV.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Guanina/análogos & derivados , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Lamivudina/química , Lamivudina/farmacologia , Nucleosídeos/análogos & derivados , Antivirais/química , Antivirais/farmacologia , Sequência de Bases , Cristalografia por Raios X , DNA Viral/química , Nucleotídeos de Desoxicitosina , Nucleotídeos de Desoxiguanina , Desenho de Fármacos , Guanina/química , Guanina/farmacologia , HIV-1/genética , Mutação/genética , Conformação de Ácido Nucleico , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia
10.
Biomed Pharmacother ; 122: 109698, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31918272

RESUMO

AIM: We aim to explore the effects of nucleos(t)ide analogues (NUCs) on the changes of HBsAg in chronic hepatitis B (CHB) patients. METHODS: A total of 264 CHB patients were enrolled in our study. All of them were treated with NUCs for at least three years. Quantification of HBsAg levels were measured by Elecsys HBsAg II. RESULTS: Although HBsAg levels were significantly higher in HBeAg seropositive CHB patients at baseline than in HBeAg seronegative CHB patients (3.84 ±â€¯0.82 vs 3.21 ±â€¯0.59 IU/mL), HBsAg levels declined more rapidly in the HBeAg seropositive group (P < 0.001). In HBeAg-positive CHB patients, HBsAg level in the telbivudine (LDT)-treated group was 3.68 ±â€¯0.56 IU/mL after 52-week of treatment, which was significantly higher than that in lamivudine (LAM)-treated group (P = 0.009). Multivariable analyses showed that baseline HBV DNA viral load (OR = 0.75, P = 0.018), baseline ALT level (OR = 0.99, P = 0.015), and baseline HBsAg level (OR = 0.188, P < 0.001) were independent factors that affected HBsAg decline in HBeAg seropositive CHB patients. For HBeAg seronegative CHB patients, the average of serum HBsAg levels in LAM-, LdT-, adefovir (ADV)-, and entecavir (ETV)-treated groups at baseline, 52 weeks, 104 weeks, and 156 weeks were similar. Multivariable analyses showed that only baseline HBV DNA level (OR = 0.56, P = 0.020) and baseline HBsAg level (OR = 0.57, P = 0.012) were independent factors that affected HBsAg decline in HBeAg seronegative patients with CHB. Baseline HBV DNA level (OR = 0.72, P = 0.010) and baseline HBsAg level (OR = 0.19, P < 0.001) were independent factors that affected all CHB patients. CONCLUSIONS: CHB Patients who had received NUCs antiviral treatment showed a slow but significant decrease in serum HBsAg level. Long-term monitoring and continuous antiviral treatment are necessary, especially for those patients with risk factors associated with HBsAg decline.


Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Nucleotidases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Adulto , DNA Viral/efeitos dos fármacos , Feminino , Guanina/análogos & derivados , Guanina/farmacologia , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Humanos , Masculino , Organofosfonatos/farmacologia , Carga Viral/efeitos dos fármacos
11.
Life Sci ; 244: 117333, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962132

RESUMO

AIMS: Detect the antiarrhythmic effect of crotonoside (Cro). MAIN METHODS: We used whole-cell patch-clamp techniques to detect the effects of Cro on action potentials (APs) and transmembrane ion currents in isolated rabbit left ventricular myocytes. We also verified the effect of Cro on ventricular arrhythmias caused by aconitine in vivo. KEY FINDINGS: Cro reduced the maximum depolarization velocity (Vmax) of APs and shortened the action potential duration (APD) in a concentration-dependent manner, but it had no significant effect on the resting membrane potential (RMP) or action potential amplitude (APA). It also inhibited the peak sodium current (INa) and L-type calcium current (ICaL) in a concentration-dependent manner with half-maximal inhibitory concentrations (IC50) of 192 µmol/L and 159 µmol/L, respectively. However, Cro had no significant effects on the inward rectifier potassium current (IK1) or rapidly activating delayed rectifier potassium current (IKr). Sea anemone toxin II (ATX II) increased the late sodium current (INaL), but Cro abolished this effect. Moreover, Cro significantly abolished ATX II-induced early afterdepolarizations (EADs) and high extracellular Ca2+ concentration (3.6 mmol/L)-induced delayed afterdepolarizations (DADs). We also verified that Cro effectively delayed the onset time and reduced the incidence of ventricular arrhythmias caused by aconitine in vivo. SIGNIFICANCE: These results revealed that Cro effectively inhibits INa, INaL, and ICaL in ventricular myocytes. Cro has antiarrhythmic potential and thus deserves further study.


Assuntos
Guanina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , China , Feminino , Guanina/metabolismo , Ventrículos do Coração/metabolismo , Técnicas de Patch-Clamp/métodos , Coelhos , Sódio/metabolismo , Canais de Sódio/efeitos dos fármacos
12.
PLoS One ; 15(1): e0227231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923260

RESUMO

A platform capable of specifically delivering an antiviral drug to the liver infected with hepatitis B is a major concern in hepatology. Vaccination has had a major effect on decreasing the emerging numbers of new cases of infection. However, the total elimination of the hepatitis B virus from the body requires prolonged therapy. In this work, we aimed to target the liver macrophages with lipid polymer hybrid nanoparticles (LPH), combining the merit of polymeric nanoparticles and lipid vesicles. The hydrophilic antiviral drug, entecavir (E), loaded LPH nanoparticles were optimized and physicochemically characterized. A modulated lipidic corona, as well as, an additional coat with vitamin E were used to extend the drug release enhance the macrophage uptake. The selected vitamin E coated LPH nanoparticles enriched with lecithin-glyceryl monostearate lipid shell exhibited high entrapment for E (80.47%), a size ≤ 200 nm for liver passive targeting, extended release over one week, proven serum stability, retained stability after refrigeration storage for 6 months. Upon macrophage uptake in vitro assessment, the presented formulation displayed promising traits, enhancing the cellular retention in J774 macrophages cells. In vivo and antiviral activity futuristic studies would help in the potential application of the ELPH in hepatitis B control.


Assuntos
Antivirais/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Guanina/análogos & derivados , Hepatite B/metabolismo , Lipídeos/química , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Polímeros/química , Vitamina E/química , Animais , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Eritrócitos/efeitos dos fármacos , Guanina/administração & dosagem , Guanina/farmacologia , Guanina/uso terapêutico , Hepatite B/tratamento farmacológico , Masculino , Camundongos , Ratos
13.
Nucleic Acids Res ; 48(4): 2107-2125, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31832686

RESUMO

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.


Assuntos
Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/química , Proteínas Ribossômicas/genética , Riboswitch/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Sítios de Ligação/genética , Escherichia coli/química , Escherichia coli/genética , Guanina/farmacologia , Ligantes , Pirimidinonas/farmacologia , Pirróis/farmacologia , RNA/efeitos dos fármacos , RNA/genética , Dobramento de RNA/efeitos dos fármacos , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/genética , Riboswitch/efeitos dos fármacos , Imagem Individual de Molécula
14.
Pharm Biol ; 58(1): 1-7, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31847670

RESUMO

Context: Fuzheng Huayu recipe (FZHY) combined with entecavir (ETV) is used to treat the cirrhosis caused by chronic hepatitis B (CHB) infection.Objective: To investigate the effect of FZHY on ETV pharmacokinetics under different conditions.Materials and methods: A model of liver fibrosis was created by intraperitoneal injection of dimethylnitrosamine (DMN; 10 µg/kg) for 4 weeks in Wistar rats. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to determine the blood concentration of ETV. Pharmacokinetic characteristics of ETV (0.9 mg/kg) were investigated after co-administration with FZHY (0.55 g/kg) at certain time intervals in normal and model rats.Results: The analytical method for ETV was validated at 0.5-50 µg/L with a correlation coefficient = 0.9996, lower limit of quantitation of 0.5 µg/L and mean accuracy of 104.18 ± 9.46%. Compared with the ETV-N group, the pharmacokinetic parameters of the EF-2 group did not change significantly, but that of the EF-0 group decreased in Cmax to 27.38 µg/L, in AUC0-t from 323.84 to 236.67 µg/h/L, and a delay in Tmax from 0.75 to 6.00 h; that of the EF-0 group presented a decrease in Cmax of 61.92%, delay in t1/2 of 2.45 h and delay in Tmax of 2.92 h. The t1/2e and Vd/F of ETV were increased significantly to 8.01 h and 24.38 L/kg in the ETV-M group.Conclusions: The effects of FZHY on ETV pharmacokinetics were diminished with an increase of interval time. The best time to administer both drugs is >2 h apart.


Assuntos
Antivirais/farmacocinética , Medicamentos de Ervas Chinesas/administração & dosagem , Guanina/análogos & derivados , Cirrose Hepática/fisiopatologia , Animais , Antivirais/administração & dosagem , Área Sob a Curva , Cromatografia Líquida de Alta Pressão/métodos , Dimetilnitrosamina , Esquema de Medicação , Medicamentos de Ervas Chinesas/farmacologia , Guanina/administração & dosagem , Guanina/farmacologia , Meia-Vida , Interações Ervas-Drogas , Masculino , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem/métodos
15.
Acta Biochim Pol ; 66(4): 585-588, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804790

RESUMO

Fluorescent tri-cyclic purine analogs, derivatives of isoguanine and adenine, were examined as potential substrates of purine-nucleoside phosphorylase. It was found previously that etheno- derivatives of both compounds are ribosylated in phosphate-free media, but ribosylation places in some instances differ from purine N9. New ribosides are examined as potential substrates of human blood PNP and indicators of this enzyme. Of these, N6-riboside of 1,N6-etheno-adenine was found the most promising.


Assuntos
Adenina/química , Glicosídeos/química , Guanina/química , Purina-Núcleosídeo Fosforilase/sangue , Adenina/farmacologia , Fluorescência , Guanina/farmacologia , Humanos , Cinética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Purinas/química
16.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569643

RESUMO

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA/química , Guanina/análogos & derivados , Mutagênicos/farmacologia , DNA Polimerase beta/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/química , Guanina/farmacologia , Halogenação , Humanos , Ligação de Hidrogênio , Cinética , Modelos Biológicos , Conformação Molecular , Mutagênicos/química
17.
World J Gastroenterol ; 25(33): 4985-4998, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543688

RESUMO

BACKGROUND: Hepatitis B virus (HBV) DNA polymerase mutations usually occur to long term use of nucleos(t)ide analogues (NAs), but they can occur spontaneously in treatment-naïve chronic hepatitis B (CHB) patients. The naturally occurring HBV DNA polymerase mutations might complicate antiviral therapy with NAs, leading to the generation of drug-resistant viral mutants and disease progression. The most common substitutions are known to be YMDD-motif mutations, but their prevalence and the influence on antiviral therapy is unclear. AIM: To investigate prevalence of the naturally occurring rtM204I mutations in treatment-naïve CHB genotype C2 patients and their influence on antiviral therapy. METHODS: A total of 410 treatment-naïve CHB patients infected with HBV genotype C2 strains were enrolled in this retrospective study. Among the 410 patients, 232 were treated with NAs for at least 12 mo. Significant fibrosis was defined as fibrosis-4 index > 3.25 or aspartate aminotransferase to platelet ratio index > 1.5. Complete viral response (CVR) during NAs was defined as undetectable serum HBV DNA (< 24 IU/mL). The rtM204I variants were analyzed by a newly developed locked nucleotide probe (LNA probe) based real-time PCR (LNA-RT-PCR) method. RESULTS: The LNA-RT-PCR could discriminate rtM204I mutant-type (17 patients, 4.2%) from rtM204 wild-type (386 patients, 95.8%) in 403 of 410 patients (98.3% sensitivity). Multivariate analysis showed that naturally occurring rtM204I variants were more frequently detected in patients with significant fibrosis [odd-ratio (OR) 3.397, 95% confidence-interval (CI) 1.119-10.319, P = 0.031]. Of 232 patients receiving NAs, multivariate analysis revealed that achievement of CVR was reversely associated with naturally occurring rtM204I variants prior to NAs treatment (OR 0.014, 95%CI 0.002-0.096, P < 0.001). Almost patients receiving tenofovir achieved CVR at 12 mo of tenofovir, irrespective of pre-existence of naturally occurring rtM204I mutations (CVR rates: patients with rtM204I, 100%; patients without rtM204I, 96.6%), whereas, pre-existence of naturally-occurring rtM204I-mutations prior to NAs significantly affects CVR rates in patients receiving entecavir (at 12 mo: Patients with rtM204I, 16.7%; patients without rtM204I, 95.6%, P < 0.001). CONCLUSION: The newly developed LNA-RT-PCR method could detect naturally occurring rtM204I mutations with high-sensitivity. Theses mutations were more frequent in patients with liver fibrosis. Tenofovir is a more suitable treatment than entecavir for CHB patients carrying the naturally occurring rtM204I mutations.


Assuntos
Produtos do Gene pol/genética , Guanina/análogos & derivados , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Tenofovir/uso terapêutico , Adulto , Análise Mutacional de DNA/métodos , Sondas de DNA/genética , DNA Viral/isolamento & purificação , Farmacorresistência Viral/genética , Estudos de Viabilidade , Feminino , Guanina/farmacologia , Guanina/uso terapêutico , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Tenofovir/farmacologia
18.
J Antibiot (Tokyo) ; 72(12): 855-864, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31554959

RESUMO

Nucleoside antibiotics possess various biological activities such as antibacterial, antifungal, anticancer, and herbicidal activities. RIKEN scientists contributed to this area of research with two representative antifungal nucleoside antibiotics, blasticidin S and polyoxin. Blasticidin S was the first antibiotic exploited in agriculture worldwide. Meanwhile, the polyoxins discovered by Isono and Suzuki are still used globally as an agricultural antibiotic. In this review article, the research on nucleoside antibiotics mainly done by Isono and his collaborators is summarized from the discovery of polyoxin to subsequent investigations.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacologia , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Azepinas/química , Azepinas/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Descoberta de Drogas , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Guanina/análogos & derivados , Guanina/química , Guanina/farmacologia , Nucleosídeos/química , Nucleosídeos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Nucleotídeos de Purina/química , Nucleotídeos de Purina/farmacologia , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/metabolismo , Nucleosídeos de Pirimidina/farmacologia , Ribonucleosídeos/química , Ribonucleosídeos/farmacologia , Uridina/análogos & derivados , Uridina/química , Uridina/farmacologia
19.
Chem Asian J ; 14(22): 3962-3968, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31389664

RESUMO

Eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of Ebola virus (EBOV) and/or other hemorrhagic fever virus (HFV) infections. They can be considered as either (i) adenine analogues (3-deazaneplanocin A, galidesivir, GS-6620 and remdesivir) or (ii) guanine analogues containing the carboxamide entity (ribavirin, EICAR, pyrazofurin and favipiravir). All eight owe their mechanism of action to hydrogen bonded base pairing with either (i) uracil or (ii) cytosine. Four out of the eight compounds (galidesivir, GS-6620, remdesivir and pyrazofurin) are C-nucleosides, and two of them (GS-6620, remdesivir) also contain a phosphoramidate part. The C-nucleoside and phosphoramidate (and for the adenine analogues the 1'-cyano group as well) may be considered as essential attributes for their antiviral activity.


Assuntos
Adenina/análogos & derivados , Antivirais/química , Guanina/análogos & derivados , Febres Hemorrágicas Virais/tratamento farmacológico , Adenina/farmacologia , Adenina/uso terapêutico , Amidas/química , Amidas/metabolismo , Amidas/farmacologia , Amidas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Pareamento de Bases , Ebolavirus/efeitos dos fármacos , Guanina/farmacologia , Guanina/uso terapêutico , Humanos , Nucleotídeos/química , Nucleotídeos/uso terapêutico , Ácidos Fosfóricos/química , Ácidos Fosfóricos/uso terapêutico , Pirazinas/química , Pirazinas/metabolismo , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Triazinas/química , Triazinas/uso terapêutico
20.
Eur J Pharm Sci ; 136: 104958, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31212018

RESUMO

Alternative formulations of entecavir, a once daily oral hepatitis B antiretroviral, may improve treatment adherence by patients. We explored the use of biocompatible polymers to control entecavir dissolution in two formats suitable for subcutaneous implantation. Hot melt extrudates were prepared by extruding entecavir-polymer blends at specified weight ratios. Dip-coated tablets were prepared by compressing entecavir in a multi-tip tooling. Tablets were dip-coated in solutions of polymer and dried. In rodents, entecavir-poly(caprolactone) extrudates demonstrated >180 days of continuous drug release, although below the estimated efficacious target input rate. Drug pharmacokinetic profiles were tunable by varying the polymer employed and implant format. The rank order trends of drug input rates observed in vitro were observed in vivo in the detected plasma concentrations of entecavir. In all dose groups entecavir was not tolerated locally at the site of administration where adverse event severity correlated with drug input rate. These polymer-based implantable formats have applicability to long-acting formulations of high solubility compounds beyond entecavir.


Assuntos
Antivirais/química , Antivirais/farmacologia , Guanina/análogos & derivados , Hepatite B/tratamento farmacológico , Animais , Química Farmacêutica/métodos , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos/efeitos dos fármacos , Excipientes/química , Feminino , Guanina/química , Guanina/farmacologia , Masculino , Polímeros/química , Ratos , Ratos Wistar , Solubilidade/efeitos dos fármacos , Comprimidos/química , Comprimidos/farmacologia
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