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1.
Cell ; 149(1): 214-31, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22464331

RESUMO

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.


Assuntos
Histonas/química , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Acetilação , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Genoma Humano , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Domínios e Motivos de Interação entre Proteínas , Proteoma/análise
2.
J Virol ; 97(10): e0039623, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37706687

RESUMO

IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.


Assuntos
Antivirais , Coronavirus , HIV-1 , Harmina , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Harmina/farmacologia , Harmina/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Replicação Viral/efeitos dos fármacos
3.
Mol Cell ; 29(5): 552-62, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18342603

RESUMO

Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and, in the S. cerevisiae lineage, this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks.


Assuntos
Evolução Molecular , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Ribossomos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Candida albicans/genética , Candida albicans/metabolismo , Biologia Computacional , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Genoma Fúngico , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Regulon , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Shelterina , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/genética
4.
Virology ; 589: 109915, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37931588

RESUMO

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.


Assuntos
Glicosídeos Cardíacos , Coronavirus Humano 229E , Humanos , Glicosídeos Cardíacos/farmacologia , Monensin/farmacologia , Ouabaína/farmacologia , Digitoxina/farmacologia , Antivirais/farmacologia
5.
Curr Protoc ; 2(1): e341, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35025140

RESUMO

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Células-Tronco Pluripotentes , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endoderma , Humanos , Pulmão , Reprodutibilidade dos Testes
6.
Nat Commun ; 12(1): 979, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579912

RESUMO

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Splicing de RNA , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Ann N Y Acad Sci ; 1082: 91-102, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17145930

RESUMO

Antisense oligonucleotides (AON) are being developed for a wide array of therapeutic applications. Significant improvements in their serum stability, target affinity, and safety profile have been achieved with the development of chemically modified oligonucleotides. Here, we compared 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)-containing AONs with phosphorothioate oligodeoxynucleotides (PS-DNA), 2'-O-methyl-RNA/DNA chimeras and short interfering RNAs (siRNA) with respect to their target knockdown efficacy, duration of action and resistance to nuclease degradation. Results show that two different configurations of FANA/DNA chimeras (altimers and gapmers) were found to have potent antisense activity. Specific target inhibition was observed with both FANA configurations with an estimated EC50 value comparable to that of an siRNA but 20-to 100-fold lower than the other commonly used AONs. Moreover, the FANA/DNA chimeras showed increased serum stability that was correlated with sustained antisense activity for up to 4 days. Taken together, these results indicate that chimeric FANA/DNA AONs are promising new tools for therapeutic gene silencing when increased potency and duration of action are required.


Assuntos
Arabinonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Estabilidade de Medicamentos , Inativação Gênica , Células HeLa , Humanos , Concentração Inibidora 50 , Luciferases/antagonistas & inibidores , Luciferases/genética , RNA Interferente Pequeno/farmacologia , Relação Estrutura-Atividade , Tionucleotídeos/farmacologia , Fatores de Tempo
8.
Methods Mol Biol ; 288: 65-80, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15333898

RESUMO

Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Primers do DNA , Oligonucleotídeos Antissenso/química , RNA/química
9.
Curr Top Med Chem ; 2(10): 1147-71, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12173972

RESUMO

A comparison of carbohydrate modified nucleic acids has identified key structural characteristics in antisense oligonucleotides (AON) that are necessary for sufficient clinical utility, including increased duplex stability towards RNA complements and improved hydrolytic resistance towards general serum and cellular nucleases. As such, the exogenous addition of short, synthetic oligonucleotides can influence cellular RNA metabolism at any or all levels of replication, transcription or translation by tight and specific hybridization with a chosen target and subsequently stop further function at that site. Furthermore, appropriate modification of the sugar residue may prove to be a vital design element in future AONs that operate by promoting enzyme assisted catalytic destruction of the mRNA target. Unfortunately, many of the current AON designs have provided little insight on the particular structural role of the AON towards enzymatic discrimination of the resultant hybrid. The use of RNase H as a cellular vehicle to assist the inhibitory potency of an AON as well as possible ways of enhancing activity in pre-existing antisense candidates are presented. Of the emerging criteria in this aspect, a balance between flexibility and rigidity within the AON appears to be a critical mediator of the RNase H assisted antisense effect. Accordingly, this review describes the conformational features and selected biological attributes of some of the more prominent AON contenders with a focus on the conformational criteria by which ribonuclease H activity is recruited to a particular hybrid target.


Assuntos
Oligonucleotídeos Antissenso/química , Pentoses/química , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Ribonuclease H/antagonistas & inibidores , Relação Estrutura-Atividade
10.
Artigo em Inglês | MEDLINE | ID: mdl-14565392

RESUMO

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2''-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2''-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.


Assuntos
Oligodesoxirribonucleotídeos/síntese química , Oligorribonucleotídeos/síntese química , Dicroísmo Circular , DNA/química , Flúor , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/química , RNA/química
11.
Nucleosides Nucleotides Nucleic Acids ; 22(5-8): 1599-602, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14565475

RESUMO

A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.


Assuntos
Splicing de RNA/genética , RNA Fúngico/química , RNA Fúngico/genética , Conformação de Ácido Nucleico , RNA Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia
12.
Curr Protoc Nucleic Acid Chem ; Chapter 4: Unit 4.15, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18428897

RESUMO

This unit describes the chemical synthesis of 2'-deoxy-2'-fluoro-b-D-oligoarabinonucleotides (2'F-ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.


Assuntos
Arabinonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Oligonucleotídeos Fosforotioatos/síntese química , Arabinose/análogos & derivados
13.
J Am Chem Soc ; 125(3): 654-61, 2003 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-12526664

RESUMO

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.


Assuntos
Arabinose/análogos & derivados , DNA Antissenso/química , Oligonucleotídeos Antissenso/química , RNA Complementar/química , Ribonuclease H/química , Arabinose/química , DNA/química , DNA/metabolismo , DNA Antissenso/síntese química , Escherichia coli/enzimologia , Genes ras , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Oligonucleotídeos Antissenso/síntese química , RNA Complementar/metabolismo , Ribonuclease H/metabolismo , Relação Estrutura-Atividade
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