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1.
Artigo em Inglês | MEDLINE | ID: mdl-35810537

RESUMO

A fully validated, simple, rapid and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine NHC (N-hydroxycytidine), the active metabolite of Molnupiravir (MOL) in human plasma; one of the limited treatment options for SARS-CoV-2 in plasma of healthy volunteers. The internal standard (IS) used was ribavirin. The extraction of analyte and IS from plasma was performed using acetonitrile as a solvent for protein precipitation. Agilent Zorbax Eclipse plus C18, 4.6 × 150 mm, (5 µm) was used for chromatographic separation using a mixture of methanol0.2 % acetic acid (5:95, v/v) as a mobile phase that was pumped at a flow rate of 0.9 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing positive ESI interface using API4500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 260.10 â†’ 128.10 and 245.10 â†’ 113.20 for NHC and IS respectively. Method validation was performed in accordance with United States FDA bioanalytical guidance. The concentration range of 20.0-10000.0 ng/mL was used to establish linearity via weighted linear regression approach (1/x2). Moreover, the analyzed pharmacokinetic data from twelve Egyptian healthy volunteers were used to develop a population pharmacokinetic model for NHC. The developed model was used to perform simulations and evaluate the current MOL dosing recommendations through calculating the maximum concentration (Cmax) "the safety metric" and area under the curve (AUC0-12 h) "the efficacy metric" for 1000 virtual subjects. Geometric mean ratios (GMR) with their associated 90% confidence intervals (CI) compared to literature values were computed. Geometric means of simulation-based Cmax and AUC0-12 were 3827 ng/mL (GMR = 1.05; 90% CI = 0.96-1.15) and 9320 ng.h/mL (GMR = 1.04; 90% CI = 0.97-1.11), respectively indicating that current MOL dosage can achieve the therapeutic targets and dose adjustment may not be required for the Egyptian population. The developed model could be used in the future to refine MOL dosage once further therapeutic targets are identified.


Assuntos
COVID-19 , Pró-Fármacos , Antivirais , Cromatografia Líquida/métodos , Citidina/análogos & derivados , Egito , Voluntários Saudáveis , Humanos , Hidroxilaminas , Reprodutibilidade dos Testes , SARS-CoV-2 , Espectrometria de Massas em Tandem/métodos
2.
Nat Commun ; 13(1): 4416, 2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906230

RESUMO

SARS-CoV-2 variants of concern (VOC) have triggered infection waves. Oral antivirals such as molnupiravir promise to improve disease management, but efficacy against VOC delta was questioned and potency against omicron is unknown. This study evaluates molnupiravir against VOC in human airway epithelium organoids, ferrets, and a lethal Roborovski dwarf hamster model of severe COVID-19-like lung injury. VOC were equally inhibited by molnupiravir in cells and organoids. Treatment reduced shedding in ferrets and prevented transmission. Pathogenicity in dwarf hamsters was VOC-dependent and highest for delta, gamma, and omicron. All molnupiravir-treated dwarf hamsters survived, showing reduction in lung virus load from one (delta) to four (gamma) orders of magnitude. Treatment effect size varied in individual dwarf hamsters infected with omicron and was significant in males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir in human trials and alerts that benefit must be reassessed in vivo as VOC evolve.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , COVID-19/tratamento farmacológico , Cricetinae , Citidina/análogos & derivados , Furões , Humanos , Hidroxilaminas , Pulmão , Masculino
3.
Genes (Basel) ; 13(7)2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35886036

RESUMO

Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated.


Assuntos
Antocianinas , Regulação da Expressão Gênica de Plantas , Citidina/análogos & derivados , Metilação de DNA/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
4.
Nat Commun ; 13(1): 4176, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853884

RESUMO

Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm5C- and f5C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m5C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f5C sites on select tRNA. Finally, we show that f5C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.


Assuntos
Dioxigenases , RNA , Homólogo AlkB 1 da Histona H2a Dioxigenase , Citidina/análogos & derivados , Dioxigenases/genética , Humanos , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
5.
Drugs Today (Barc) ; 58(7): 335-350, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35851869

RESUMO

Molnupiravir (MK-4482, EIDD-2801) is a promising broad-spectrum experimental antiviral developed by Merck & Co. It is a nucleoside analogue prodrug that undergoes rapid conversion into nucleoside triphosphate (NTP) by intracellular metabolic processes. NTP inhibits viral polymerase by acting as an alternative substrate. Molnupiravir was initially developed to treat influenza and Venezuelan equine encephalitis virus (VEEV) infection as it exerts its antiviral activity by inhibiting RNA-dependent RNA polymerase (RdRp). Currently, it is being developed for the treatment of SARS-CoV-2 infection. Molnupiravir has demonstrated potent in vitro antiviral activity against positive-sense RNA viruses including influenza viruses, SARS-CoV, SARS-CoV-2 and MERS-CoV with low cytotoxicity and a high resistance barrier. Molnupiravir has been evaluated in phase I, II and III trials where it has demonstrated good efficacy, dose-dependent pharmacokinetics and a sound safety profile. In an interim analysis of a phase III study, treatment with molnupiravir reduced the risk of hospitalization or death by 50% in patients with COVID-19; in the final analysis, the reduction was 30%. On the basis of positive results in clinical trials, molnupiravir has been authorized for emergency use by the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and the U.S. Food and Drug Administration (FDA) in adults with mild to moderate COVID-19.


Assuntos
COVID-19 , Antivirais/efeitos adversos , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Humanos , Hidroxilaminas , SARS-CoV-2 , Estados Unidos
6.
Pharmacoeconomics ; 40(7): 699-714, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35779197

RESUMO

BACKGROUND AND AIMS: Coronavirus disease 2019 (COVID-19) imposes a substantial and ongoing burden on the US healthcare system and society. Molnupiravir is a new oral antiviral for treating COVID-19 in outpatient settings. This study evaluated the cost-effectiveness profile of molnupiravir versus best supportive care in the treatment of adult patients with mild-to-moderate COVID-19 at risk of progression to severe disease, from a US payer's perspective. METHODS: The model was developed using a decision tree for the short-term acute phase of COVID-19 and a Markov state transition model for the long-term post-acute phase. This model compared molnupiravir with best supportive care as consistent with the MOVe-OUT trial. Costs were reported in 2021 US dollars. Transition probabilities were derived from the phase III MOVe-OUT trial and the TriNetX real-world electronic health records database. Costs were derived from the TriNetX database and utility values from a de novo, vignette-based utility study. Deterministic and probabilistic sensitivity analyses (DSA/PSA) were conducted. Primary outcomes included proportion hospitalized, proportion who died overall and by highest healthcare setting at the end of the acute phase, quality-adjusted life-years (QALYs), and incremental costs per QALY gained over a lifetime (100 years) horizon, discounted at 3% annually and assessed at a willingness-to-pay (WTP) threshold of $100,000 per QALY. RESULTS: In this model, the use of molnupiravir led to an increase in QALYs (0.210) and decrease in direct total medical costs (-$895) per patient across a lifetime horizon, compared with best supportive care in COVID-19 outpatients. Molnupiravir was the dominant intervention when compared with best supportive care. Patients treated with molnupiravir were less likely to be hospitalized (6.38% vs. 9.20%) and more likely to remain alive (99.88% vs. 98.71%) during the acute phase. Through DSA, molnupiravir treatment effect of hospitalization reduction was identified to be the most influential parameter, and through PSA, molnupiravir remained dominant in 84% of the total simulations and, overall, 100% cost effective. CONCLUSION: This analysis suggests that molnupiravir is cost effective compared with best supportive care for the treatment of adult outpatients with COVID-19. However, our study was limited by the unavailability of the most recent information on the rapidly evolving pandemic, including new viral variants, patient populations affected, and changes in standards of care. Further research should explore the impact of vaccination on the cost effectiveness of molnupiravir and other therapies, based on real-world data, to account for these changes, including the impact of vaccination and immunity.


Assuntos
COVID-19 , Pacientes Ambulatoriais , Adulto , Análise Custo-Benefício , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Masculino , Antígeno Prostático Específico , Anos de Vida Ajustados por Qualidade de Vida
7.
Tissue Cell ; 77: 101850, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35679684

RESUMO

Wnt/ß-catenin, a highly conserved signaling pathway, is involved in determining cell fate. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiac cells. This study is aimed to investigate the role of Wnt/ß-catenin signaling in cardiac lineage commitment of human umbilical cord mesenchymal stem cells (hUCMSCs) after treatment with demethylating agents, zebularine and 2'-deoxycytidine (2-DC). hUCMSCs were treated with 20 µM zebularine or 2-DC for 24 h and cultured for 14 days. Control and treated MSCs were analyzed for cardiac lineage commitment at gene and protein levels. Significant upregulation of early and late cardiac markers, GATA4, Nkx2.5, cardiac myosin heavy chain (cMHC), α-actinin, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) was observed in treated MSCs as compared to the untreated control. We also analyzed gene expression of key Wnt/ß-catenin signaling molecules in cultures of treated and untreated hUCMSCs at 24 h, and days 3, 7 and 14. The pattern of mRNA gene expression showed that Wnt/ß-catenin signaling is regulated during cardiac lineage commitment of hUCMSCs in a time-dependent manner, with the pathway being activated early but inhibited later in cardiac development. Findings of this study can lead us to identify more specific and effective strategies for cardiac lineage commitment.


Assuntos
Células-Tronco Mesenquimais , beta Catenina , Diferenciação Celular , Citidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Miócitos Cardíacos/metabolismo , Cordão Umbilical , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
8.
Mol Cell ; 82(15): 2797-2814.e11, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35679869

RESUMO

mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. Although cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5' UTRs impacts protein synthesis at the level of initiation. 5' UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed that ac4C in the -1 position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner.


Assuntos
Citidina , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Animais , Códon de Iniciação , Citidina/análogos & derivados , Citidina/genética , Mamíferos/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Front Immunol ; 13: 914577, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35757739

RESUMO

Background: 5-Methylcytidine (m5C) methylation is an emerging epigenetic modification in recent years, which is associated with the development and progression of various cancers. However, the prognostic value of m5C regulatory genes and the correlation between m5C methylation and the tumor microenvironment (TME) in prostate cancer remain unknown. Methods: In the current study, the genetic and transcriptional alterations and prognostic value of m5C regulatory genes were investigated in The Cancer Genome Atlas and Gene Expression Omnibus datasets. Then, an m5C prognostic model was established by LASSO Cox regression analysis. Gene set variation analyses (GSVA), gene set enrichment analysis (GSEA), clinical relevance, and TME analyses were conducted to explain the biological functions and quantify the TME scores between high-risk and low-risk subgroups. m5C regulatory gene clusters and m5C immune subtypes were identified using consensus unsupervised clustering analysis. The Cell-type Identification By Estimating Relative Subsets of RNA Transcripts algorithm was used to calculate the contents of immune cells. Results: TET3 was upregulated at transcriptional levels in PCa compared with normal tissues, and a high TET3 expression was associated with poor prognosis. An m5C prognostic model consisting of 3 genes (NSUN2, TET3, and YBX1) was developed and a nomogram was constructed for improving the clinical applicability of the model. Functional analysis revealed the enrichment of pathways and the biological processes associated with RNA regulation and immune function. Significant differences were also found in the expression levels of m5C regulatory genes, TME scores, and immune cell infiltration levels between different risk subgroups. We identified two distinct m5C gene clusters and found their correlation with patient prognosis and immune cell infiltration characteristics. Naive B cells, CD8+ T cells, M1 macrophages and M2 macrophages were obtained and 2 m5C immune subtypes were identified. CTLA4, NSUN6, TET1, and TET3 were differentially expressed between immune subtypes. The expression of CTLA4 was found to be correlated with the degree of immune cell infiltration. Conclusions: Our comprehensive analysis of m5C regulatory genes in PCa demonstrated their potential roles in the prognosis, clinical features, and TME. These findings may improve our understanding of m5C regulatory genes in the tumor biology of PCa.


Assuntos
Citidina/análogos & derivados , Neoplasias da Próstata , Citidina/genética , Citidina/metabolismo , Genes Reguladores , Humanos , Masculino , Metilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA/genética , RNA/metabolismo , Microambiente Tumoral/genética
10.
Viruses ; 14(6)2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35746614

RESUMO

Enterovirus infections can cause hand, foot, and mouth disease (HFDM), aseptic meningitis, encephalitis, myocarditis, and acute flaccid myelitis, leading to death of infants and young children. However, no specific antiviral drug is currently available for the treatment of this type of infection. The Unites States and United Kingdom health authorities recently approved a new antiviral drug, molnupiravir, for the treatment of COVID-19. In this study, we reported that molnupiravir (EIDD-2801) and its active form, EIDD-1931, have broad-spectrum anti-enterovirus potential. Our data showed that EIDD-1931 could significantly reduce the production of EV-A71 progeny virus and the expression of EV-A71 viral protein at non-cytotoxic concentrations. The results of the time-of-addition assay suggest that EIDD-1931 acts at the post-entry step, which is in accordance with its antiviral mechanism. The intraperitoneal administration of EIDD-1931 and EIDD-2801 protected 1-day-old ICR suckling mice from lethal EV-A71 challenge by reducing the viral load in various tissues of the infected mice. The pharmacokinetics analysis indicated that the plasma drug concentration overwhelmed the EC50 for enteroviruses, suggesting the clinical potential of molnupiravir against enteroviruses. Thus, molnupiravir along with its active form, EIDD-1931, may be a promising drug candidate against enterovirus infections.


Assuntos
COVID-19 , Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Animais , Antígenos Virais/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Pré-Escolar , Citidina/análogos & derivados , Enterovirus/metabolismo , Infecções por Enterovirus/tratamento farmacológico , Humanos , Hidroxilaminas , Camundongos , Camundongos Endogâmicos ICR
11.
Viruses ; 14(6)2022 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-35746815

RESUMO

Molnupiravir is a ß-d-N4-hydroxycytidine-5'-isopropyl ester (NHC) compound that exerts antiviral activity against various RNA viruses such as influenza, SARS, and Ebola viruses. Thus, the repurposing of Molnupiravir has gained significant attention for combatting infection with SARS-CoV-2, the etiological agent of COVID-19. Recently, Molnupiravir was granted authorization for the treatment of mild-to-moderate COVID-19 in adults. Findings from in vitro experiments, in vivo studies and clinical trials reveal that Molnupiravir is effective against SARS-CoV-2 by inducing viral RNA mutagenesis, thereby giving rise to mutated complementary RNA strands that generate non-functional viruses. To date, the data collectively suggest that Molnupiravir possesses promising antiviral activity as well as favorable prophylactic efficacy, attributed to its effective mutagenic property of disrupting viral replication. This review discusses the mechanisms of action of Molnupiravir and highlights its clinical utility by disabling SARS-CoV-2 replication, thereby ameliorating COVID-19 severity. Despite relatively few short-term adverse effects thus far, further detailed clinical studies and long-term pharmacovigilance are needed in view of its mutagenic effects.


Assuntos
COVID-19 , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Humanos , Hidroxilaminas , SARS-CoV-2
12.
Biomed Pharmacother ; 150: 113058, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35658229

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new variants and generally short duration of immunity, the development of effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing drugs for accelerated clinical testing and emergency use authorizations. However, drug-repurposing studies using cellular assays often identify hits that later prove ineffective clinically, highlighting the need for more complex screening models. To this end, we evaluated the activity of single compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Furthermore, we also evaluated drug combinations based on a sub-maximal concentration of molnupiravir. We report the antiviral activity of 95 single compounds and 30 combinations. We show that only a few single agents are highly effective in inhibiting SARS-CoV-2 replication while selected drug combinations containing 10 µM molnupiravir boosted antiviral activity compared to single compound treatment. These data indicate that molnupiravir-based combinations are worthy of further consideration as potential treatment strategies against coronavirus disease 2019 (COVID-19).


Assuntos
COVID-19 , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Mucosa Nasal , SARS-CoV-2
14.
Plant J ; 111(3): 756-767, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35652245

RESUMO

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.


Assuntos
Bryopsida , Edição de RNA , Bryopsida/genética , Citidina/metabolismo , Nucleotídeos/genética , Nucleotídeos/metabolismo , Proteínas de Plantas/metabolismo , Edição de RNA/genética , RNA de Plantas/metabolismo
15.
Biol Pharm Bull ; 45(6): 770-779, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650104

RESUMO

Neuronal cell death after cerebral ischemia consists various steps including glutamate excitotoxity. Excessive Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, which is one of the ionotropic glutamate receptors, plays a central role in neuronal cell death after cerebral ischemia. We previously reported that DNA methylation is transiently increased in neurons during ischemic injury and that this aberrant DNA methylation is accompanied by neuronal cell death. Therefore, we performed the present experiments on glutamate excitotoxicity to gain further insight into DNA methylation involvement in the neuronal cell death. We demonstrated that knockdown of DNA methyltransferase (DNMT)1, DNMT3a, or DNMT3b gene in Neuro2a cells was performed to examine which DNMTs were more important for neuronal cell death after glutamate excitotoxicity. Although we confirmed a decrease in the levels of the target DNMT protein after small interfering RNA (siRNA) transfection, the Neuro2a cells were not protected from injury by transfection with siRNA for each DNMT. We next revealed that the pharmacological inhibitor of DNMTs protected against glutamate excitotoxicity in Neuro2a cells and also in primary cultured cortical neurons. This protective effect was associated with a decrease in the number of 5-methylcytosine (5 mC)-positive cells under glutamate excitotoxicity. In addition, the increased level of cleaved caspase-3 was also reduced by a DNMT inhibitor. Our results suggest the possibility that at least 2 or all DNMTs functionally would cooperate to activate DNA methylation after glutamate excitotoxicity and that inhibition of DNA methylation in neurons after cerebral ischemia might become a strategy to reduce the neuronal injury.


Assuntos
Isquemia Encefálica , Ácido Glutâmico , Morte Celular , Citidina/análogos & derivados , Metilação de DNA , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Humanos , RNA Interferente Pequeno/genética , Receptores de N-Metil-D-Aspartato/metabolismo
16.
JCI Insight ; 7(13)2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35579953

RESUMO

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC), which contains a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here, we assessed the efficacy of MK-4482 against the earlier Alpha, Beta, and Delta VOCs and Omicron in the hamster COVID-19 model. Omicron replication and associated lung disease in vehicle-treated hamsters was reduced compared with replication and lung disease associated with earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of hamsters infected with Alpha, Beta, or Delta VOCs. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious titers compared with viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , COVID-19/tratamento farmacológico , Cricetinae , Citidina/análogos & derivados , Humanos , Hidroxilaminas
17.
Biochem J ; 479(11): 1149-1164, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35583288

RESUMO

Uridine-cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein with high sequence similarity to other uridine-cytidine kinases (UCKs). UCKs play an important role in the pyrimidine salvage pathway, catalyzing the phosphorylation of uridine and cytidine to UMP and CMP, respectively. Only two human UCKs have been identified, UCK1 and UCK2. Previous studies have shown both enzymes phosphorylate uridine and cytidine using ATP as the phosphate donor. No studies have evaluated the kinase potential of UCKL-1. We cloned and purified UCKL-1 and found that it successfully phosphorylated uridine and cytidine using ATP as the phosphate donor. The catalytic efficiency (calculated as kcat/KM) was 1.2 × 104 s-1, M-1 for uridine and 0.7 × 104 s-1, M-1 for cytidine. Our lab has previously shown that UCKL-1 is up-regulated in tumor cells, providing protection against natural killer (NK) cell killing activity. We utilized small interfering RNA (siRNA) to down-regulate UCKL-1 in vitro and in vivo to determine the effect of UCKL-1 on tumor growth and metastasis. The down-regulation of UCKL-1 in YAC-1 lymphoma cells in vitro resulted in decreased cell counts and increased apoptotic activity. Down-regulation of UCKL-1 in K562 leukemia cells in vivo led to decreased primary tumor growth and less tumor cell dissemination and metastasis. These results identify UCKL-1 as a bona fide pyrimidine kinase with the therapeutic potential to be a target for tumor growth inhibition and for diminishing or preventing metastasis.


Assuntos
Citidina , Uridina Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina/genética , Citidina/metabolismo , Citidina/farmacologia , Humanos , Fosfatos , Fosforilação , Fosfotransferases , Pirimidinas/metabolismo , RNA Interferente Pequeno/metabolismo , Uridina/metabolismo , Uridina Quinase/genética
18.
Biotechnol Lett ; 44(7): 831-843, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35608787

RESUMO

PURPOSE: In the wake of SARS-CoV-2's global spread, human activities from health to social life to education have been affected. Favipiravir and Molnupiravir exhibited novel hexokinase inhibition and we discuss advantages of this property in their COVID-19 inhibition potential. METHODS: This paper describes molecular docking data of human hexokinase II with Favipiravir, Cyan 20, Remdesivir, 2DG, and Molnupiravir along with hexokinase inhibition assays. RESULTS: Favipiravir, an antiviral drug previously cleared for treating the flu and ebola, has shown some promise in early trials to treat COVID-19. We observed potent human hexokinase inhibiting potential of Favipiravir (50%) as against 4% and merely 0.3% hexokinase inhibition with Molnupiravir and 2 Deoxy D glucose at 0.1 mM concentration supported by molecular docking studies. CONCLUSION: Favipiravir could continue to be part of the COVID-19 treatment regimen due to its resistance to host esterases, hexokinase inhibition potential and proven safety through human trials.


Assuntos
COVID-19 , Amidas , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Desoxiglucose/farmacologia , Hexoquinase , Humanos , Hidroxilaminas , Simulação de Acoplamento Molecular , Pirazinas , SARS-CoV-2
19.
Clin Transl Med ; 12(5): e738, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35522942

RESUMO

BACKGROUND: Dysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA translation efficiency through RNA acetylation. However, the role of NAT10-mediated acetylation modification in bladder cancer remains elusive. METHODS: The clinical value of NAT10 was estimated according to NAT10 expression pattern based on TCGA data set and the tumor tissue array. Acetylated RNA immunoprecipitation sequencing was utilized to explore the role of NAT10 in mRNA ac4C modification. Translation efficiency and mRNA stability assay were applied to study the effect of NAT10-deletion on target genes. The nude mouse model and genetically engineered mice were conducted to further verify the characteristics of NAT10 in promoting BLCA progression and regulating downstream targets. RESULTS: NAT10 was essential for the proliferation, migration, invasion, survival and the stem-cell-like properties of bladder cancer cell lines. NAT10 was responsible for mRNA ac4C modification in BLCA cells, including BCL9L, SOX4 and AKT1. Deficient NAT10 in both xenograft and transgenic mouse models of bladder cancer reduced the tumor burden. Furthermore, acetylated RNA immunoprecipitation sequencing data and RNA immunoprecipitation qPCR results revealed that NAT10 is responsible for a set of ac4C mRNA modifications in bladder cancer cells. Inhibition of NAT10 led to a loss of ac4C peaks in these transcripts and represses the mRNA's stability and protein expression. Mechanistically, the ac4C reduction modification in specific regions of mRNAs resulting from NAT10 downregulation impaired the translation efficiency of BCL9L, SOX4 and AKT1 as well as the stability of BCL9L, SOX4. CONCLUSIONS: In summary, these findings provide new insights into the dynamic characteristics of mRNA's post-transcriptional modification via NAT10-dependent acetylation and predict a role for NAT10 as a therapeutic target in bladder cancer. HIGHLIGHTS: NAT10 is highly expressed in BLCA patients and its abnormal level predicts bladder cancer progression and low overall survival rate. NAT10 is necessary and sufficient for BLCA tumourigenic properties. NAT10 is responsible for ac4C modification of target transcripts, including BCL9L, SOX4 and AKT1. NAT10 may serve as an effective and novel therapeutic target for BLCA.


Assuntos
Acetiltransferases N-Terminal , Neoplasias da Bexiga Urinária , Animais , Citidina/análogos & derivados , Citidina/genética , Humanos , Camundongos , Acetiltransferases N-Terminal/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXC , Neoplasias da Bexiga Urinária/genética
20.
J Pharm Sci ; 111(8): 2201-2209, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35526576

RESUMO

Methoxy trityl groups are acid-responsive protecting groups that are routinely used in the process of nucleoside analog synthesis. This study investigated the potential of methoxy trityl groups, monomethoxy trityl (MMT), dimethoxy trityl (DMT), and trimethoxy trityl (TMT), as acid-responsive substituents for designing anti-cancer cytidine analog prodrugs. For this purpose, we synthesized six gemcitabine (GEM) derivatives, which were modified either 4-(N)- or 5'-(O)-sites with MMT, DMT, and TMT, as candidates for anti-cancer cytidine analog prodrugs. In vitro dissociation test of methoxy trityl groups clearly showed that the acid responsivity of the methoxy trityl moieties was in the order TMT>DMT>MMT. Furthermore, the rate of 5'-(O)-methoxy tritylation was higher than that of 4-(N)-methoxy tritylation. Along with high acid-responsivity, trimethoxy trityl-O-GEM (TMT-O-GEM) showed superior cytotoxicity against 2D cultured human breast cancer cells (MCF-7 and MDA-MB-231) and human pancreatic cancer cells (AsPC-1) compared to other methoxy-tritylated GEM derivatives. Moreover, TMT-O-GEM suppressed the growth of MCF-7 spheroids compared with trimethoxy trityl-N-GEM (TMT-N-GEM). Both TMT-O-GEM and TMT-N-GEM were negligibly deprotected and metabolized in mouse or human serum after 72 h, indicating that trimethoxy tritylation inhibits deamination by cytidine deaminase. These results indicate that 5'-(O)-trimethoxy tritylation is a potent approach for the development of anti-cancer cytidine analog prodrugs.


Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Pró-Fármacos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citidina/farmacologia , Citidina/uso terapêutico , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico
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