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1.
Med Sci Monit ; 28: e935952, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34972812

RESUMO

On 4th November 2021, the first oral antiviral drug for COVID-19, molnupiravir (Lagevrio®), received full regulatory approval from the Medicines and Healthcare Products Regulatory Agency (MHRA) in the UK. Molnupiravir is an orally bioavailable antiviral drug for use at home when a SARS-CoV-2 test is positive. On 22nd December 2022, the FDA granted emergency use authorization (EUA) for the oral antiviral drug, nirmatrelvir/ritonavir (Paxlovid®) for adults and children with mild and moderate COVID-19 at increased risk of progression to severe COVID-19. These regulatory drug approvals come at a crucial time when new variants of concern of the SARS-CoV-2 virus are spreading rapidly. Although the FDA approved remdesivir (Veklury®) on 22nd October 2020 for use in adults and children for the treatment of COVID-19 requiring hospitalization, its use has been limited by the requirement for intravenous administration in a healthcare facility. The four FDA-approved therapeutic neutralizing monoclonal antibodies, imdevimab, bamlanivimab, etesevimab, and casirivimab are costly and also require medically-supervised intravenous administration. The availability of effective, low-cost oral antiviral drugs available in a community setting that can be used at an early stage of SARS-CoV-2 infection is now a priority in controlling COVID-19. An increasing number of repurposed antiviral drugs are currently under investigation or in the early stages of regulatory approval. This Editorial aims to present an update on the current status of orally bioavailable antiviral drug treatments for SARS-CoV-2 infection.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Hidroxilaminas/uso terapêutico , Administração Oral , Anticorpos Monoclonais/uso terapêutico , Citidina/uso terapêutico , Aprovação de Drogas , Reposicionamento de Medicamentos/tendências , Humanos , Lactamas/uso terapêutico , Leucina/uso terapêutico , Nitrilas/uso terapêutico , Prolina/uso terapêutico , Ritonavir/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Estados Unidos , United States Food and Drug Administration
2.
Pharmacol Res Perspect ; 10(1): e00909, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34968008

RESUMO

The novel coronavirus disease 2019 (COVID-19) emerged in late December 2019 in china and has rapidly spread to many countries around the world. The effective pharmacotherapy can reduce the mortality of COVID-19. Antiviral medications are the candidate therapies for the management of COVID-19. Molnupiravir is an antiviral drug with anti-RNA polymerase activity and currently is under investigation for the treatment of patients with COVID-19. This review focuses on summarizing published literature for the mechanism of action, safety, efficacy, and clinical trials of molnupiravir in the treatment of COVID-19 patients.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Hidroxilaminas/uso terapêutico , COVID-19/virologia , Ensaios Clínicos como Assunto , Citidina/uso terapêutico , Interações Medicamentosas , Humanos , SARS-CoV-2/isolamento & purificação
3.
Methods Mol Biol ; 2404: 363-374, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694620

RESUMO

Recent studies have uncovered that cellular mRNAs contain a diverse epitranscriptome comprising chemically modified bases which play important roles in gene expression regulation. Among these is m6A, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for m6A profiling have used m6A antibodies to immunoprecipitate methylated RNAs. Although powerful, such methods require high amounts of input material. Recently, we developed DART-seq, an antibody-free method for m6A profiling from low-input RNA samples. DART-seq relies on deamination of cytidines that invariably follow m6A sites and can be performed using a simple in vitro assay with only 50 ng of total RNA. Here, we describe the in vitro DART method and present a detailed protocol for highly sensitive m6A profiling from any RNA sample of interest.


Assuntos
RNA/genética , Análise de Sequência de RNA , Citidina , Regulação da Expressão Gênica
5.
Int J Med Sci ; 18(16): 3788-3793, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34790054

RESUMO

As the world is racing to develop perpetual immunity to the SARS-CoV-2 virus. The emergence of new viral strains, together with vaccination and reinfections, are all contributing to a long-term immunity against the deadly virus that has taken over the world since its introduction to humans in late December 2019. The discovery that more than 95 percent of people who recovered from COVID-19 had long-lasting immunity and that asymptomatic people have a different immune response to SARS-CoV-2 than symptomatic people has shifted attention to how our immune system initiates such diverse responses. These findings have provided reason to believe that SARS-CoV-2 days are numbered. Hundreds of research papers have been published on the causes of long-lasting immune responses and variations in the numbers of different immune cell types in COVID 19 survivors, but the main reason of these differences has still not been adequately identified. In this article, we focus on the activation-induced cytidine deaminase (AID), which initiates molecular processes that allow our immune system to generate antibodies against SARS-CoV-2. To establish lasting immunity to SARS-CoV-2, we suggest that AID could be the key to unlocking it.


Assuntos
COVID-19/imunologia , Citidina Desaminase/genética , Imunidade/genética , SARS-CoV-2/imunologia , COVID-19/virologia , Citidina/genética , Citidina/imunologia , Citidina Desaminase/imunologia , Desaminação/imunologia , Humanos , SARS-CoV-2/patogenicidade , Vacinação
7.
Curr Protoc ; 1(11): e295, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34792862

RESUMO

This article provides a detailed procedure for the chemical synthesis and characterization of photoswitchable hydrazone phosphoramidite and its incorporation into oligodeoxynucleotides. The synthesis starts with commercially available deoxyuridine, followed by conversion of the 4-oxo into a 4-chloro moiety via Appel reaction to install the key hydrazone group in the absence of base. The hydrazone phosphoramidite building block is compatible with the conventional amidite chemistry protocols for solid-phase synthesis of oligodeoxynucleotides. Our method expands the current nucleotide pool by adding a novel, functional DNA building block that is suitable for a broad spectrum of applications, including the regulation of DNA-enzyme interactions and DNA synthesis by irradiation with cell-friendly blue light. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of photoswitchable hydrazone phosphoramidite Basic Protocol 2: Synthesis and purification of oligodeoxynucleotides containing the hydrazone photoswitch Basic Protocol 3: Primer extension assay for functionality studies of hydrazone cytidine.


Assuntos
Hidrazonas , Oligodesoxirribonucleotídeos , Citidina , DNA , Técnicas de Síntese em Fase Sólida
8.
Curr Protoc ; 1(11): e307, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34792865

RESUMO

This protocol describes a step-by-step chemical synthesis approach to prepare N3 -methylcytidine (m3 C) and its phosphoramidite. The method for synthesizing m3 C starts from commercially available cytidine, and proceeds via N3 -methylation in the presence of MeI, which generates the N3 -methylcytidine (m3 C) nucleoside, followed by the installation of several protecting groups at sites that include the 5'-hydroxyl group (4,4'-dimethoxytrityl protection), the 4-amino group (benzoyl protection), and the 2'-hydroxyl group (tert-butyldimethylsilyl, TBDMS, protection). Standard phosphoramidite chemistry is applied to prepare the final m3 C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N3 -methylcytidine (m3 C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m3 C modified RNA oligonucleotides.


Assuntos
Oligonucleotídeos , RNA , Citidina , Nucleosídeos , Técnicas de Síntese em Fase Sólida
11.
Virology ; 564: 33-38, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34619630

RESUMO

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.


Assuntos
Coronavirus Humano 229E/genética , Infecções por Coronavirus/tratamento farmacológico , Coronavirus Humano NL63/genética , Coronavirus Humano OC43/genética , Citidina/análogos & derivados , Hidroxilaminas/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Resfriado Comum/tratamento farmacológico , Coronavirus Humano 229E/efeitos dos fármacos , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/efeitos dos fármacos , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus Humano OC43/fisiologia , Citidina/farmacologia , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Pirrolidinas/farmacologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Estações do Ano , Ácidos Sulfônicos/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
13.
Molecules ; 26(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34641339

RESUMO

The COVID-19 pandemic needs no introduction at present. Only a few treatments are available for this disease, including remdesivir and favipiravir. Accordingly, the pharmaceutical industry is striving to develop new treatments for COVID-19. Molnupiravir, an orally active RdRp inhibitor, is in a phase 3 clinical trial against COVID-19. The objective of this review article is to enlighten the researchers working on COVID-19 about the discovery, recent developments, and patents related to molnupiravir. Molnupiravir was originally developed for the treatment of influenza at Emory University, USA. However, this drug has also demonstrated activity against a variety of viruses, including SARS-CoV-2. Now it is being jointly developed by Emory University, Ridgeback Biotherapeutics, and Merck to treat COVID-19. The published clinical data indicate a good safety profile, tolerability, and oral bioavailability of molnupiravir in humans. The patient-compliant oral dosage form of molnupiravir may hit the market in the first or second quarter of 2022. The patent data of molnupiravir revealed its granted compound patent and process-related patent applications. We also anticipate patent filing related to oral dosage forms, inhalers, and a combination of molnupiravir with marketed drugs like remdesivir, favipiravir, and baricitinib. The current pandemic demands a patient compliant, safe, tolerable, and orally effective COVID-19 treatment. The authors believe that molnupiravir meets these requirements and is a breakthrough COVID-19 treatment.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Citidina/análogos & derivados , Descoberta de Drogas , Hidroxilaminas/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/química , Ensaios Clínicos como Assunto , Citidina/administração & dosagem , Citidina/química , Citidina/uso terapêutico , Humanos , Hidroxilaminas/administração & dosagem , Hidroxilaminas/química , Patentes como Assunto , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/uso terapêutico , SARS-CoV-2/enzimologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo
15.
Mol Pharmacol ; 100(6): 548-557, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34503974

RESUMO

Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, ß-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 µM; ENT2 IC50: 77 µM), followed by EIDD-1931 (ENT1 IC50: 259 µM; ENT2 IC50: 467 µM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 µM; ENT2 IC50: 851 µM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/metabolismo , Citidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , SARS-CoV-2/metabolismo , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Alanina/metabolismo , Antivirais/administração & dosagem , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Citidina/administração & dosagem , Citidina/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , SARS-CoV-2/efeitos dos fármacos
16.
J Pharm Biomed Anal ; 206: 114356, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34509661

RESUMO

In light of the recent global pandemic, Molnupiravir (MPV) or EIDD-2801, developed for the treatment of patients with uncomplicated influenza, is now being trialled for the treatment of infections caused by highly pathogenic coronaviruses, including COVID-19. A sensitive LC-MS/MS method was developed and validated for the simultaneous quantification of MPV and its metabolite ß-d-N4-hydroxycytidine (NHC) in human plasma and saliva. The analytes were extracted from the matrices by protein precipitation using acetonitrile. This was followed by drying and subsequently injecting the reconstituted solutions onto the column. Chromatographic separation was achieved using a polar Atlantis C18 column with gradient elution of 1 mM Ammonium acetate in water (pH4.3) and 1 mM Ammonium acetate in acetonitrile. Analyte detection was conducted in negative ionisation mode using SRM. Analysis was performed using stable isotopically labelled (SIL) internal standards (IS). The m/z transitions were: MPV (328.1→126.0), NHC (258.0→125.9) and MPV-SIL (331.0→129.0), NHC-SIL (260.9→128.9). Validation was over a linear range of 2.5-5000 ng/ml for both plasma and saliva. Across four different concentrations, precision and accuracy (intra- and inter-day) were 15%; and recovery of both analytes from plasma and saliva was between 95% and 100% and 65-86% respectively. Clinical pharmacokinetic studies are underway utilising this method for determination of MPV and its metabolite in patients with COVID-19 infection.


Assuntos
COVID-19 , Saliva , Cromatografia Líquida , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Reprodutibilidade dos Testes , SARS-CoV-2 , Espectrometria de Massas em Tandem
17.
Artigo em Inglês | MEDLINE | ID: mdl-34555541

RESUMO

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to ß-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. ß-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1-5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1-1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.


Assuntos
Citidina/análogos & derivados , Citidina/sangue , Citidina/química , Humanos , Reprodutibilidade dos Testes
18.
J Biotechnol ; 341: 129-136, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34536458

RESUMO

Industrial production of neuroprotective drug CDP-choline is accomplished via permeabilized or lysed cell biotransformation because of the inefficient penetration of substrates into intact cells. We previously proposed a novel one-step living cell method for CDP-choline production by engineered yeast, but obtained low titer and molar yield. This study develops a high-production strain with improved molar yield by metabolic engineering strategies. The selective markers previously integrated into host cell were recovered for facilitating genetic modification, which however resulted a strain with improved CDP-choline titer and molar yield to CMP. Knockout of 5'-NT or CDA in CMP sinking pathway but not APY in CTP sinking pathway further improved CDP-choline titer and molar yield to CMP. However, overexpression of seven enzymes in CTP synthetic pathway showed no positive functions. Finally, optimization of CMP and choline phosphate levels for the optimized recombinant strains achieved a high-level CDP-choline of ~30 g/L, which was enhanced by 400% compared to the previous work. Also, the molar yield of CDP-choline to CMP increased from 40% to 84.7%. The titer and molar yield are comparable to the reported permeabilized or lysed cell based biotransformation methods. It represents a novel and competitive paradigm for the potential industrial production of CDP-choline.


Assuntos
Citidina Difosfato Colina , Saccharomyces cerevisiae , Citidina , Engenharia Metabólica , Saccharomyces cerevisiae/genética
19.
Methods Enzymol ; 658: 25-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517949

RESUMO

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven ß-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of m7G, m3C, D, and ho5C in variety of cellular RNAs, including in particular rRNAs and tRNAs.


Assuntos
Citidina , Guanosina , Citidina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA de Transferência/genética , Análise de Sequência de RNA
20.
Curr Protoc ; 1(9): e248, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34529364

RESUMO

This article summarizes the protocols for phosphoramidite chemistry and solid phase synthesis of RNA oligonucleotides containing N4 -methylcytidine (m4 C) and N4 ,N4 -dimethylcytidine (m4 2 C) residues for base-pairing, structural, and enzymatic activity studies. The two key m4 C and m4 2 C phosphoramidite building blocks can be synthesized starting from the partially protected cytidine nucleosides, followed by solid-phase synthesis and HPLC purification of the modified target RNA oligonucleotides. These modified RNA strands are then prepared for base pairing stability, specificity, and structural studies using UV-melting temperature (Tm ) measurements and X-ray crystallography. Functional studies are performed by reverse transcription assays in primer extension reactions employing different enzymes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of m4 C phosphoramidite Basic Protocol 2: Synthesis of m4 2 C phosphoramidite Basic Protocol 3: Synthesis and purification of m4 C and m4 2 C containing RNA oligonucleotides.


Assuntos
Citidina , RNA , Pareamento de Bases , Nucleosídeos , Oligonucleotídeos
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