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1.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
2.
ACS Chem Biol ; 16(7): 1234-1242, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34192867

RESUMO

Understanding the selectivity of methyltransferase inhibitors is important to dissecting the functions of each methyltransferase target. From this perspective, we report a chemoproteomic study to profile the selectivity of a potent protein N-terminal methyltransferase 1 (NTMT1) bisubstrate inhibitor NAH-C3-GPKK (Ki, app = 7 ± 1 nM) in endogenous proteomes. First, we describe the rational design, synthesis, and biochemical characterization of a new chemical probe 6, a biotinylated analogue of NAH-C3-GPKK. Next, we systematically analyze protein networks that may selectively interact with the biotinylated probe 6 in concert with the competitor NAH-C3-GPKK. Besides NTMT1, the designated NTMT1 bisubstrate inhibitor NAH-C3-GPKK was found to also potently inhibit a methyltransferase complex HemK2-Trm112 (also known as KMT9-Trm112), highlighting the importance of systematic selectivity profiling. Furthermore, this is the first potent inhibitor for HemK2/KMT9 reported until now. Thus, our studies lay the foundation for future efforts to develop selective inhibitors for either methyltransferase.


Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Oligopeptídeos/farmacologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Adenosina/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Metiltransferases/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ligação Proteica
3.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198328

RESUMO

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismo
4.
Antiviral Res ; 193: 105142, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34303749

RESUMO

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 µM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Nucleotidiltransferases/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , COVID-19/virologia , Linhagem Celular , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Metiltransferases/metabolismo , Nucleotidiltransferases/metabolismo , Capuzes de RNA , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
7.
Molecules ; 26(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800013

RESUMO

With the emergence and global spread of the COVID-19 pandemic, the scientific community worldwide has focused on search for new therapeutic strategies against this disease. One such critical approach is targeting proteins such as helicases that regulate most of the SARS-CoV-2 RNA metabolism. The purpose of the current study was to predict a library of phytochemicals derived from diverse plant families with high binding affinity to SARS-CoV-2 helicase (Nsp13) enzyme. High throughput virtual screening of the Medicinal Plant Database for Drug Design (MPD3) database was performed on SARS-CoV-2 helicase using AutoDock Vina. Nilotinib, with a docking value of -9.6 kcal/mol, was chosen as a reference molecule. A compound (PubChem CID: 110143421, ZINC database ID: ZINC257223845, eMolecules: 43290531) was screened as the best binder (binding energy of -10.2 kcal/mol on average) to the enzyme by using repeated docking runs in the screening process. On inspection, the compound was disclosed to show different binding sites of the triangular pockets collectively formed by Rec1A, Rec2A, and 1B domains and a stalk domain at the base. The molecule is often bound to the ATP binding site (referred to as binding site 2) of the helicase enzyme. The compound was further discovered to fulfill drug-likeness and lead-likeness criteria, have good physicochemical and pharmacokinetics properties, and to be non-toxic. Molecular dynamic simulation analysis of the control/lead compound complexes demonstrated the formation of stable complexes with good intermolecular binding affinity. Lastly, affirmation of the docking simulation studies was accomplished by estimating the binding free energy by MMPB/GBSA technique. Taken together, these findings present further in silco investigation of plant-derived lead compounds to effectively address COVID-19.


Assuntos
Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacocinética , Antivirais/toxicidade , Sítios de Ligação , Disponibilidade Biológica , COVID-19/tratamento farmacológico , Biologia Computacional/métodos , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Metiltransferases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Plantas Medicinais/química , Ligação Proteica , Domínios Proteicos/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/toxicidade , RNA Helicases/química , Relação Estrutura-Atividade , Termodinâmica , Proteínas não Estruturais Virais/química
8.
Theranostics ; 11(8): 3676-3693, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33664855

RESUMO

Background: Methyltransferase-like 14 (METTL14) participates in tumorigenesis in several malignancies, but how METTL14 mediates the metastasis of renal cell carcinoma (RCC) has never been reported. Methods: Western blotting, quantitative real-time PCR, and immunohistochemistry were used to determine the mRNA and protein levels of relevant genes. Methylated RNA immunoprecipitation sequencing and RNA sequencing were utilized to screen potential targets of METTL14. Chromatin immunoprecipitation sequencing and assay for transposase-accessible chromatin sequencing were performed to investigate epigenetic alterations. The biological roles and mechanisms of METTL14/BPTF in promoting lung metastasis were confirmed in vitro and in vivo using cell lines, patient samples, xenograft models, and organoids. Results: Utilizing the TCGA-KIRC and Ruijin-RCC datasets, we found low expression of METTL14 in mRCC samples, which predicted poor prognosis. METTL14 deficiency promoted RCC metastasis in vitro and in vivo. Mechanistically, METTL14-mediated m6A modification negatively regulated the mRNA stability of bromodomain PHD finger transcription factor (BPTF) and depended on BPTF to drive lung metastasis. Accumulated BPTF in METTL14-deficient cells remodeled the enhancer landscape to reinforce several oncogenic crosstalk. Particularly, BPTF constituted super-enhancers that activate downstream targets like enolase 2 and SRC proto-oncogene nonreceptor tyrosine kinase, leading to glycolytic reprogramming of METTL14-/- cells. Finally, we determined the efficacy of the BPTF inhibitor AU1 in suppressing mRCC of patient-derived cells, mRCC-derived organoids (MDOs), and orthotopic xenograft models. Conclusions: Our study is the first to investigate the essential role of m6A modification and the METTL14/BPTF axis in the epigenetic and metabolic remodeling of mRCC, highlighting AU1 as a vital therapeutic candidate.


Assuntos
Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adamantano/análogos & derivados , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/secundário , Linhagem Celular Tumoral , Regulação para Baixo , Elementos Facilitadores Genéticos , Técnicas de Inativação de Genes , Glicólise , Humanos , Neoplasias Renais/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Metiltransferases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/antagonistas & inibidores , Compostos de Fenilureia , Medicina de Precisão , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Expert Opin Ther Pat ; 31(4): 339-350, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33593200

RESUMO

Introduction: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC50 of patented inhibitors ranges between 0.82 µM and 8.95 µM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.Areas covered: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.Expert opinion: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Desenvolvimento de Medicamentos , Metiltransferases/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Humanos , Metiltransferases/química , Metiltransferases/fisiologia , Patentes como Assunto , RNA Helicases/química , RNA Helicases/fisiologia , Triazóis/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/fisiologia
10.
J Med Chem ; 64(12): 7900-7925, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33599482

RESUMO

Epigenetic regulation of gene expression plays a critical role in various physiological processes, and epigenetic dysregulation is implicated in a number of diseases, prominently including cancer. Epigenetic regulators have been validated as potential therapeutic targets, and significant progress has been made in the discovery and development of epigenetic-based inhibitors. However, successful epigenetic drug discovery is still facing challenges, including moderate selectivity, limited efficacy, and acquired drug resistance. Inspired by the advantages of covalent small-molecule inhibitors, targeted covalent inhibition has attracted increasing interest in epigenetic drug discovery. In this review, we comprehensively summarize the structure-based design and characterization of covalent inhibitors targeting epigenetic writers, readers, and erasers and highlight their potential benefits in enhancing selectivity across the enzyme family and improving in vivo efficacy. We also discuss the challenges and opportunities of covalent small-molecule inhibitors and hope to shed light on future epigenetic drug discovery.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Ligação Proteica , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo
11.
PLoS One ; 16(2): e0246181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33596235

RESUMO

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Metiltransferases/metabolismo , RNA Helicases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Sítio Alostérico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Biologia Computacional/métodos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/química , Modelos Moleculares , Terapia de Alvo Molecular , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
12.
Anal Chem ; 93(4): 2403-2410, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33395263

RESUMO

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning HM with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.


Assuntos
Azacitidina/farmacologia , Fluoruracila/farmacologia , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Antimetabólitos/farmacologia , Escherichia coli/metabolismo , Metiltransferases/química
13.
J Hazard Mater ; 408: 124490, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33199140

RESUMO

A number of epigenetic modulating chemicals are known to affect multiple generations of a population from a single ancestral exposure, thus posing transgenerational hazards. The present study aimed to establish a high-throughput (HT) analytical workflow for cost-efficient concentration-response analysis of epigenetic and phenotypic effects, and to support the development of novel Adverse Outcome Pathway (AOP) networks for DNA methyltransferase (DNMT) inhibitor-mediated transgenerational effects on aquatic organisms. The model DNMT inhibitor 5-azacytidine (5AC) and the model freshwater crustacean Daphnia magna were used to generate new experimental data and served as prototypes to construct AOPs for aquatic organisms. Targeted HT bioassays (DNMT ELISA, MS-HRM and qPCR) in combination with multigenerational ecotoxicity tests revealed concentration-dependent transgenerational (F0-F3) effects of 5AC on total DNMT activity, DNA promoter methylation, gene body methylation, gene transcription and reproduction. Top sensitive toxicity pathways related to 5AC exposure, such as apoptosis and DNA damage responses were identified in both F0 and F3 using Gaussian Bayesian network modeling. Two novel epigenetic AOP networks on DNMT inhibitor mediated one-generational and transgenerational effects were developed for aquatic organisms and assessed for the weight of evidence. The new HT analytical workflow and AOPs can facilitate future ecological hazard assessment of epigenetic modulating chemicals.


Assuntos
Rotas de Resultados Adversos , Metilação de DNA , Epigênese Genética , Metiltransferases/antagonistas & inibidores , Animais , Teorema de Bayes , DNA , Daphnia
14.
Cancer Res ; 80(22): 4998-5010, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33023947

RESUMO

Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. SIGNIFICANCE: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4998/F1.large.jpg.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Metilação de DNA , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia Tumoral/fisiologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ilhas de CpG , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citosina/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Genes erbB-1 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Sistema de Sinalização das MAP Quinases , Metiltransferases/antagonistas & inibidores , Camundongos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/fisiologia
15.
Immunogenetics ; 72(8): 423-430, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33009922

RESUMO

We have previously demonstrated that Mettl3-silencing dendritic cells (DCs) exhibited immature properties and prolonged allograft survival in a murine heart transplantation model. Exosomes derived from donor DCs (Dex) are involved in the immune rejection of organ transplantation, and blocking Dex transfer may suppress immune rejection. Herein, this study aimed to investigate whether Mettl3 knockdown inhibits the secretion and activity of donor Dex, thereby inhibiting donor Dex-mediated immune rejection. The imDex, mDex, shCtrl-mDex, and shMettl3-mDex were obtained from the culture supernatant of DCs (immature DCs, mature DCs, shCtrl-infected mature DCs, shMettl3-infected mature DCs) derived from donor BALB/c mouse bone marrow and then co-cultured with splenic T cell lymphocyte suspension from recipient C57BL/6 mice in vitro or injected into recipient C57BL/6 mice before the cardiac transplantation. Donor shMettl3-mDex expressed lower concentration of exosomes and lower expression of Mettl3, Dex markers (ICAM-1, MHC-I, MHC-II), as well as lower ability to activate T cell immune response than shCtrl-mDex. Administration of donor shMettl3-mDex attenuated immune rejection after mouse heart transplantation and prolonged the allograft survival. In summary, Mettl3 knockdown inhibits the immune rejection of Dex in a mouse cardiac allograft model.


Assuntos
Células Dendríticas/citologia , Exossomos/metabolismo , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/efeitos adversos , Tolerância Imunológica/imunologia , Metiltransferases/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Aloenxertos , Animais , Técnicas de Silenciamento de Genes , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Masculino , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
16.
Int J Biol Macromol ; 163: 1687-1696, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980406

RESUMO

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Simulação por Computador , Infecções por Coronavirus/virologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Ácido Fólico/farmacologia , Genoma Viral , Glucosídeos/farmacologia , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mutação , Pandemias , Pneumonia Viral/virologia , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2 , Estilbenos/farmacologia , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
17.
Eur J Med Chem ; 208: 112835, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32977201

RESUMO

The high mortality rate and increasing prevalence of resistant Mtb are the major concerns for the Tuberculosis (TB) treatment in this century. To curtail the prevalence of resistant Mtb, we have prepared 1,3-oxazine-2-one based dual targeted molecules. Compound 67 and 68 were found to be equally active against replicating and non-replicatiing form of Mtb (MICMABA 3.48 and 2.97 µg/ml; MICLORA 2.94 and 2.15 µg/ml respectively). They had found to suppress the biosynthesis of alfa, methoxy and keto-mycolate completely, as well as inhibit enzymatic activity of MenG (IC50 = 9.11 and 6.25 µg/ml respectively for H37Ra; IC50 = 11.76 and 10.88 µg/ml respectively for M smegmatis).


Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazinas/farmacologia , Antituberculosos/síntese química , Antituberculosos/toxicidade , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Leucócitos Mononucleares/efeitos dos fármacos , Metiltransferases/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Oxazinas/síntese química , Oxazinas/toxicidade , Relação Estrutura-Atividade
18.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , COVID-19 , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , SARS-CoV-2 , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
19.
Life Sci ; 259: 118169, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738360

RESUMO

AIMS: The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. MATERIALS AND METHODS: The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2'OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2'OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2'OMTase followed by re-docking analysis. The apo and ligand-bound 2'OMTase were further validated and analyzed by using molecular dynamics simulation. KEY FINDINGS: The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2'OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2'OMTase. SIGNIFICANCE: The results suggested that these drugs may be used as potential inhibitors for 2'OMTase for combating the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , COVID-19 , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Homologia de Sequência de Aminoácidos
20.
PLoS One ; 15(8): e0236882, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790688

RESUMO

N6-methyladenosine (m6A) is the most prevalent type of RNA modification. METTL3 in the methyltransferase complex is the core enzyme responsible for methylation. METTL3 selectively catalyzes the adenosines centered in the RRAC motif. Functional studies established that m6A could enhance the translation efficiency (TE) of modified genes by recruiting reader protein YTHDF1 and other initiation factors. We downloaded the m6A peaks in HeLa cells from a previous study and defined the m6A modified genes and sites. Ancestral mutations in the genic region fixed in the HeLa cell samples were defined using their mRNA-Seq data and the alignment between human and mouse genomes. Furthermore, in the small interfering (si)-METTL3 sample, the calculated TE foldchange of all genes was compared to that in the negative control. The TE of m6A genes was globally down-regulated in si-METTL3 versus control compared to the non-m6A genes. In m6A modified genes, RRAC motif mutations were suppressed compared to mutations in non-motif regions or non-m6A genes. Among the m6A genes, a fraction RRAC motif mutations negatively correlated with the TE foldchange (si-METTL3 versus control). The TE of m6A modified genes was enhanced in HeLa cells. RRAC motif mutations could potentially prevent methylation of adenosines and consequently abolish the enhanced translation. Such mutations in the RRAC motif might be deleterious. Accordingly, we observed lower fractions of mutations in RRAC motifs than in other regions. This prevention of mutations in the RRAC motif could be a strategy adopted by cancer cells to maintain the elevated translation of particular genes.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Sequência de Bases , Células HeLa , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Mutação , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
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