Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.713
Filtrar
1.
Sci Rep ; 11(1): 19998, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620963

RESUMO

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.


Assuntos
Antivirais/metabolismo , COVID-19/tratamento farmacológico , Descoberta de Drogas , SARS-CoV-2/efeitos dos fármacos , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/metabolismo , Adenina/farmacologia , Adenosina/efeitos adversos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Monofosfato de Adenosina/efeitos adversos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/efeitos adversos , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Amidas/efeitos adversos , Amidas/metabolismo , Amidas/farmacologia , Antivirais/efeitos adversos , Antivirais/farmacologia , COVID-19/metabolismo , Cloroquina/efeitos adversos , Cloroquina/análogos & derivados , Cloroquina/metabolismo , Cloroquina/farmacologia , Desenho de Fármacos , Humanos , Redes e Vias Metabólicas , Simulação de Acoplamento Molecular , Nitrocompostos/efeitos adversos , Nitrocompostos/metabolismo , Nitrocompostos/farmacologia , Pirazinas/efeitos adversos , Pirazinas/metabolismo , Pirazinas/farmacologia , Pirrolidinas/efeitos adversos , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ribavirina/efeitos adversos , Ribavirina/metabolismo , Ribavirina/farmacologia , SARS-CoV-2/metabolismo , Tiazóis/efeitos adversos , Tiazóis/metabolismo , Tiazóis/farmacologia
2.
Phys Chem Chem Phys ; 23(40): 23005-23013, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34611693

RESUMO

Core ionization of DNA begins a cascade of events which could lead to cellular inactivation or death. The created core-hole following an impulse inner-shell ionization of molecules naturally decays in the auger timescale. We simulated charge migration (CM) phenomena following an impulsive core ionization of individual DNA bases at the oxygen K-edge which occurs before Auger decay of the oxygen. Our approach is based on real-time time dependent density functional theory (RT-TDDFT). It is shown that the pronounced hole fluctuation observed around bonds of the initial core-hole results in various valence orbital migrations. Also, the same photo-core-ionized dynamics is studied for the related base pairs. We investigate the role of base pairing and H-bonding interactions in the attosecond CM dynamics. In particular, the creation of a core-hole in the oxygen involved in H-bonding leads to an enhancement of charge migration relative to the respective single bases. Importantly, the hole oscillation of the adenine-thymine base pair upon creation of a core-hole at the oxygen, which does not contribute to the donor-acceptor interactions (not H-bonded), decreases compared to the single thymine base. Understanding the detailed dynamics of the localized core-hole initiating CM process would open the way for chemically controlling DNA damage/repair in the future.


Assuntos
DNA/química , Oxigênio/química , Adenina/química , Adenina/metabolismo , Pareamento de Bases , DNA/metabolismo , Teoria da Densidade Funcional , Ligação de Hidrogênio , Íons/química , Timina/química , Timina/metabolismo
3.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502079

RESUMO

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules.


Assuntos
Amiloide/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Adenina/química , Adenina/metabolismo , Amiloide/química , Amiloide/metabolismo , Dimetil Sulfóxido/análogos & derivados , Simulação de Dinâmica Molecular , Polimerização/efeitos dos fármacos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
ACS Chem Biol ; 16(9): 1644-1653, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34397208

RESUMO

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.


Assuntos
Acrilamidas/química , Adenina/análogos & derivados , Afatinib/química , Compostos de Anilina/química , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Lisina/química , Inibidores de Fosfoinositídeo-3 Quinase/química , Piperidinas/química , Acetilação , Acrilamidas/metabolismo , Adenina/química , Adenina/metabolismo , Afatinib/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/metabolismo , Catálise , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
5.
Nat Commun ; 12(1): 5055, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417448

RESUMO

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) µ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol µ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol µ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol µ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Ribonucleotídeos/metabolismo , Adenina/metabolismo , Cálcio/metabolismo , Domínio Catalítico , Citosina/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Cinética , Manganês/metabolismo , Modelos Moleculares , Oxirredução
7.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209188

RESUMO

Coronavirus disease (COVID)-19 is the leading global health threat to date caused by a severe acute respiratory syndrome coronavirus (SARS-CoV-2). Recent clinical trials reported that the use of Bruton's tyrosine kinase (BTK) inhibitors to treat COVID-19 patients could reduce dyspnea and hypoxia, thromboinflammation, hypercoagulability and improve oxygenation. However, the mechanism of action remains unclear. Thus, this study employs structure-based virtual screening (SBVS) to repurpose BTK inhibitors acalabrutinib, dasatinib, evobrutinib, fostamatinib, ibrutinib, inositol 1,3,4,5-tetrakisphosphate, spebrutinib, XL418 and zanubrutinib against SARS-CoV-2. Molecular docking is conducted with BTK inhibitors against structural and nonstructural proteins of SARS-CoV-2 and host targets (ACE2, TMPRSS2 and BTK). Molecular mechanics-generalized Born surface area (MM/GBSA) calculations and molecular dynamics (MD) simulations are then carried out on the selected complexes with high binding energy. Ibrutinib and zanubrutinib are found to be the most potent of the drugs screened based on the results of computational studies. Results further show that ibrutinib and zanubrutinib could exploit different mechanisms at the viral entry and replication stage and could be repurposed as potential inhibitors of SARS-CoV-2 pathogenesis.


Assuntos
Adenina/análogos & derivados , Reposicionamento de Medicamentos , Simulação de Dinâmica Molecular , Piperidinas/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Adenina/química , Adenina/metabolismo , Adenina/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Humanos , Simulação de Acoplamento Molecular , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
8.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064609

RESUMO

Monogenic hypertension is rare and caused by genetic mutations, but whether factors associated with mutations are disease-specific remains uncertain. Given two factors associated with high mutation rates, we tested how many previously known genes match with (i) proximity to telomeres or (ii) high adenine and thymine content in cardiovascular diseases (CVDs) related to vascular stiffening. We extracted genomic information using a genome data viewer. In human chromosomes, 64 of 79 genetic loci involving >25 rare mutations and single nucleotide polymorphisms satisfied (i) or (ii), resulting in an 81% matching rate. However, this high matching rate was no longer observed as we checked the two factors in genes associated with essential hypertension (EH), thoracic aortic aneurysm (TAA), and congenital heart disease (CHD), resulting in matching rates of 53%, 70%, and 75%, respectively. A matching of telomere proximity or high adenine and thymine content projects the list of loci involving rare mutations of monogenic hypertension better than those of other CVDs, likely due to adoption of rigorous criteria for true-positive signals. Our data suggest that the factor-disease matching rate is an accurate tool that can explain deleterious mutations of monogenic hypertension at a >80% match-unlike the relatively lower matching rates found in human genes of EH, TAA, CHD, and familial Parkinson's disease.


Assuntos
Doenças Cardiovasculares/genética , Predisposição Genética para Doença , Mutação , Doenças do Sistema Nervoso/genética , Polimorfismo de Nucleotídeo Único , Telômero/genética , Adenina/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Humanos , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Timina/metabolismo
9.
Nat Commun ; 12(1): 3436, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103525

RESUMO

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.


Assuntos
Adenina/metabolismo , Clostridioides/enzimologia , DNA Bacteriano/química , Conformação de Ácido Nucleico , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Pareamento de Bases , Sequência de Bases , Coenzimas/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , S-Adenosil-Homocisteína/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade da Espécie , Especificidade por Substrato
10.
FASEB J ; 35(7): e21684, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34159634

RESUMO

Adenine nucleotides represent crucial immunomodulators in the extracellular environment. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thus promoting an anti-inflammatory milieu induced by the "adenosine halo". AMPD2 intracellularly mediates AMP deamination to IMP, thereby both enhancing the degradation of inflammatory ATP and reducing the formation of anti-inflammatory adenosine. Here, we show that this enzyme is expressed on the surface of human immune cells and its predominance may modify inflammatory states by altering the extracellular milieu. Surface AMPD2 (eAMPD2) expression on monocytes was verified by immunoblot, surface biotinylation, mass spectrometry, and immunofluorescence microscopy. Flow cytometry revealed enhanced monocytic eAMPD2 expression after TLR stimulation. PBMCs from patients with rheumatoid arthritis displayed significantly higher levels of eAMPD2 expression compared with healthy controls. Furthermore, the product of AMPD2-IMP-exerted anti-inflammatory effects, while the levels of extracellular adenosine were not impaired by an increased eAMPD2 expression. In summary, our study identifies eAMPD2 as a novel regulator of the extracellular ATP-adenosine balance adding to the immunomodulatory CD39-CD73 system.


Assuntos
5'-Nucleotidase/metabolismo , AMP Desaminase/metabolismo , Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Leucócitos/metabolismo , Apirase , Células Cultivadas , Proteínas Ligadas por GPI/metabolismo , Humanos
11.
Chemphyschem ; 22(15): 1622-1630, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34101319

RESUMO

DFT calculations are employed to quantify the influence of the presence, number, nature, and position of posttranscriptional methylation on stacking strength of RNA bases. We carry out detailed potential energy scans of the variation in stacking energies with characteristic geometrical parameters in three categories of forty stacked dimers - canonical base homodimers (N||N), methylated base homodimers (mN||mN) and heterodimers of canonical bases and methylated counterparts (N||mN). Our analysis reveals that neutral methylation invariably enhances the stacking of bases. Further, N||mN stacking is stronger than mN||mN stacking and charged N||mN exhibit strongest stacking among all dimers. This indicates that methylations greatly enhance stacking when dispersed in RNA sequences containing identical bases. Comparison of stacks involving singly- and doubly-methylated purines reveal that incremental methylation enhances the stacking in neutral dimers. Although methylation at the carbon position of neutral pyrimidine dimers greatly enhances the stacking, methylation on the 5-membered ring imparts better stacking compared to methylation on the 6-membered ring in adenine dimers. However, methylation at the ring nitrogen (N1 ) provides better stacking than the amino group (N2 ) in guanine dimers. Our results thus highlight subtle structural effects of methylation on RNA base stacking and will enhance our understanding of the physicochemical principles of RNA structure and dynamics.


Assuntos
Processamento Pós-Transcricional do RNA , RNA/química , RNA/metabolismo , Adenina/química , Adenina/metabolismo , Citosina/química , Citosina/metabolismo , Teoria da Densidade Funcional , Dimerização , Guanina/química , Guanina/metabolismo , Metilação , Uracila/química , Uracila/metabolismo
12.
Sci Rep ; 11(1): 11985, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099830

RESUMO

Recent studies suggested that ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, developed for the treatment of chronic lymphocytic leukemia, may prevent NLRP3 inflammasome activation in macrophages, IL-1ß secretion and subsequent development of inflammation and organ fibrosis. The role of NLRP3 has been underlined in the various causes of acute kidney injury (AKI), a pathology characterized by high morbimortality and risk of transition toward chronic kidney disease (CKD). We therefore hypothesized that the BTK-inhibitor ibrutinib could be a candidate drug for AKI treatment. Here, we observed in both an AKI model (glycerol-induced rhabdomyolysis) and a model of rapidly progressive kidney fibrosis (unilateral ureteral obstruction), that ibrutinib did not prevent inflammatory cell recruitment in the kidney and fibrosis. Moreover, ibrutinib pre-exposure led to high mortality rate owing to severer rhabdomyolysis and AKI. In vitro, ibrutinib potentiated or had no effect on the secretion of IL-1ß by monocytes exposed to uromodulin or myoglobin, two danger-associated molecule patterns proteins involved in the AKI to CKD transition. According to these results, ibrutinib should not be considered a candidate drug for patients developing AKI.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Adenina/análogos & derivados , Antineoplásicos/farmacologia , Fibrose/prevenção & controle , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperidinas/farmacologia , Injúria Renal Aguda/complicações , Adenina/metabolismo , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Coleta de Amostras Sanguíneas , Humanos , Rim , Leucócitos Mononucleares , Macrófagos , Masculino , Camundongos Endogâmicos C57BL , Mortalidade , Mioglobina/metabolismo , Preparações Farmacêuticas , Piperidinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Rabdomiólise/complicações
13.
Nature ; 595(7866): 295-302, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34079130

RESUMO

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.


Assuntos
Adenina/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/terapia , Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Globinas beta/genética , Animais , Antígenos CD34/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Modelos Animais de Doenças , Feminino , Terapia Genética , Genoma Humano/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Camundongos
14.
Nat Metab ; 3(5): 651-664, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972798

RESUMO

Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Purinas/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Fatores de Transcrição/metabolismo , Adenina/metabolismo , Vias Biossintéticas , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana Transportadoras , Modelos Biológicos , Proteínas Carreadoras de Solutos/genética , Fatores de Transcrição/antagonistas & inibidores , Transcrição Genética
15.
Nature ; 593(7859): 429-434, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34012082

RESUMO

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.


Assuntos
Sistemas CRISPR-Cas , LDL-Colesterol/sangue , Edição de Genes , Modelos Animais , Pró-Proteína Convertase 9/genética , Adenina/metabolismo , Animais , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Mutação com Perda de Função , Macaca fascicularis/sangue , Macaca fascicularis/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/metabolismo , Fatores de Tempo
16.
Sci Rep ; 11(1): 10357, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990665

RESUMO

DNA N6-methylation (6mA) in Adenine nucleotide is a post replication modification responsible for many biological functions. Automated and accurate computational methods can help to identify 6mA sites in long genomes saving significant time and money. Our study develops a convolutional neural network (CNN) based tool i6mA-CNN capable of identifying 6mA sites in the rice genome. Our model coordinates among multiple types of features such as PseAAC (Pseudo Amino Acid Composition) inspired customized feature vector, multiple one hot representations and dinucleotide physicochemical properties. It achieves auROC (area under Receiver Operating Characteristic curve) score of 0.98 with an overall accuracy of 93.97% using fivefold cross validation on benchmark dataset. Finally, we evaluate our model on three other plant genome 6mA site identification test datasets. Results suggest that our proposed tool is able to generalize its ability of 6mA site identification on plant genomes irrespective of plant species. An algorithm for potential motif extraction and a feature importance analysis procedure are two by products of this research. Web tool for this research can be found at: https://cutt.ly/dgp3QTR .


Assuntos
Epigenômica/métodos , Genoma de Planta , Redes Neurais de Computação , Oryza/genética , Adenina/análogos & derivados , Adenina/análise , Adenina/metabolismo , Motivos de Aminoácidos/genética , Metilação de DNA , Epigênese Genética
17.
Toxins (Basel) ; 13(4)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810228

RESUMO

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were Km of 3.14 µM and Kcat 2107 min-1. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.


Assuntos
Adenina/metabolismo , DNA/metabolismo , Técnicas Eletroquímicas , Ricina/metabolismo , Espermatozoides/metabolismo , Animais , Peixes , Cinética , Masculino , Reprodutibilidade dos Testes , Especificidade por Substrato
18.
Toxicol Appl Pharmacol ; 421: 115536, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33865896

RESUMO

Cadmium (Cd) can induce ovarian injury by microRNAs (miRNAs), however, the molecular mechanism of miRNAs after Cd exposure have not known. In this study, 56-day-old adult female Sprague-Dawley (SD) rats were injection with PMSG, after 48 h, ovarian granulosa cells (GCs) were extracted and cultured for 24 h, then treated with 0, 2.5, 5, 10 and 20 µM Cd for 24 h. The results showed that expression levels of miR-92a-2-5p (upregulated) and Bcl2 (downregulated) changed significantly after Cd exposure. The messenger RNA (mRNA) and protein expression levels of DNMT1, DNMT3A, and DNMT3B had changed, but no obvious differences were found in miR-92a-2-5p single site methylation. The transcription factors C-MYC (upregulated), E2F1 (downregulated), and SP1 (downregulated), which target miRNAs significantly changed after exposure to Cd. The human ovarian GC tumor line (COV434) was used to knocked down C-myc, and the expression of miR-92a-2-5p was downregulated in the COV434-C-myc + 10 µM Cd group compared with COV434 cells. The N6-methyladenosine (m6A) methylation modification levels of long noncoding RNA (lncRNA) MT1JP and lncRNA CDKN2B-AS, which regulate miR-92a-2-5p were detected. In the 10 µM Cd group, m6A methylation levels at MT1JP-84, CDKN2B-AS-257, and CDKN2B-AS-329 were reduced. In summary, after Cd exposure, expression of miR-92a-2-5p, which targets the antiapoptotic gene Bcl2, was upregulated, which may be primarily related to upregulation of C-myc. MiR-92a-2-5p promoter DNA methylation may has no obvious effect on miR-92a-2-5p. Otherwise, the role of m6A methylation modified lncRNA MT1JP and lncRNA CDKN2B-AS in the regulation of miR-92a-2-5p needs further study.


Assuntos
Cloreto de Cádmio/toxicidade , Células da Granulosa/efeitos dos fármacos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Feminino , Células da Granulosa/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , MicroRNAs/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos Sprague-Dawley , Regulação para Cima
19.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
20.
Nat Commun ; 12(1): 2420, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893297

RESUMO

Bacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host's restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.


Assuntos
2-Aminopurina/análogos & derivados , Adenina/química , Bacteriófagos/genética , Cianobactérias/virologia , DNA Viral/química , Synechococcus/virologia , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Adenina/metabolismo , Bacteriófagos/metabolismo , Sítios de Ligação/genética , Biocatálise , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...