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1.
Nucleic Acids Res ; 49(20): 11560-11574, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34718736

RESUMO

Friedreich's ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5' or 3' noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.


Assuntos
Ataxia de Friedreich/terapia , Terapia Genética/métodos , Proteínas de Ligação ao Ferro/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Células Cultivadas , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Frataxina
2.
Proc Natl Acad Sci U S A ; 114(8): E1509-E1518, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28193854

RESUMO

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.


Assuntos
Atrofia Muscular Espinal/terapia , Oligonucleotídeos/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Éxons/genética , Fibroblastos , Dosagem de Genes , Terapia Genética/métodos , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Mutação Puntual , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Ativação Transcricional/genética , Regulação para Cima
3.
Nucleic Acids Res ; 43(6): 2993-3011, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25753666

RESUMO

The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.


Assuntos
RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Animais , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Sítios de Ligação , LDL-Colesterol/sangue , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Fosfatos/química , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicerídeos/sangue
4.
J Biol Chem ; 290(4): 2069-85, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25505270

RESUMO

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.


Assuntos
Regulação da Expressão Gênica , Hipertensão Pulmonar/metabolismo , MicroRNAs/metabolismo , Vasoconstrição , Algoritmos , Animais , Células Cultivadas , Endotelina-1/metabolismo , Endotelinas/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Oligonucleotídeos/química , PPAR gama/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais , Biologia de Sistemas
5.
RNA ; 18(8): 1510-26, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22740638

RESUMO

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.


Assuntos
Fígado/metabolismo , MicroRNAs/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Western Blotting , Perfilação da Expressão Gênica , Células HeLa , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Polirribossomos/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA/genética , RNA Mensageiro/antagonistas & inibidores
7.
Nucleic Acids Res ; 38(20): 7298-307, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20624819

RESUMO

A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2'-fluoro or 2'-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2'-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2'-F/2'-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson-Crick base pairs with additional 3' UU overhangs and a 5' phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.


Assuntos
RNA Interferente Pequeno/química , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Prótons , Soluções
8.
J Am Chem Soc ; 133(41): 16642-9, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-21919455

RESUMO

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.


Assuntos
Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , Álcoois Açúcares/química , Modelos Moleculares , Conformação Molecular , Estereoisomerismo
9.
Nucleic Acids Res ; 37(1): 70-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19015151

RESUMO

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2'-fluoro/2'-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.


Assuntos
MicroRNAs/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Regulação da Expressão Gênica , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/análise , MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/química , Ácidos Nucleicos Peptídicos/química
10.
Biomaterials ; 275: 120966, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34147715

RESUMO

Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.


Assuntos
Nanopartículas , Polímeros , Animais , DNA , Lipídeos , Pulmão , Camundongos , RNA Mensageiro/genética , Transfecção
11.
J Org Chem ; 75(5): 1569-81, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20136157

RESUMO

We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.


Assuntos
Cristalinas/síntese química , Ácidos Nucleicos/síntese química , Nucleosídeos/síntese química , Oligonucleotídeos/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Fenômenos Biofísicos , Catálise , Cristalinas/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Ácidos Nucleicos/química , Nucleosídeos/química , Estereoisomerismo
12.
J Med Chem ; 51(9): 2766-76, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18399648

RESUMO

A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.


Assuntos
Oligorribonucleotídeos Antissenso/síntese química , Animais , Linhagem Celular , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico , Oligorribonucleotídeos Antissenso/química , Oligorribonucleotídeos Antissenso/farmacologia , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/biossíntese , Relação Estrutura-Atividade
13.
Nucleic Acids Res ; 34(16): 4467-76, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16945958

RESUMO

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Humanos , Cinesinas/genética , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo
15.
J Med Chem ; 49(5): 1624-34, 2006 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-16509579

RESUMO

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Ribonucleases/sangue , Tionucleosídeos/síntese química , Animais , Estabilidade de Medicamentos , Células HeLa , Calefação , Humanos , Técnicas In Vitro , Camundongos , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Plasma , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Ribose/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfóxidos/química , Tionucleosídeos/química , Tionucleosídeos/farmacologia
17.
J Med Chem ; 48(13): 4247-53, 2005 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-15974578

RESUMO

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.


Assuntos
Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Linhagem Celular , Mutação em Linhagem Germinativa , Células HeLa , Humanos , Mamíferos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Interferência de RNA/efeitos dos fármacos , RNA Antissenso/química , RNA Antissenso/farmacologia , RNA Neoplásico/efeitos dos fármacos , RNA Interferente Pequeno/química , Proteínas Supressoras de Tumor/antagonistas & inibidores
18.
J Med Chem ; 48(4): 901-4, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15715458

RESUMO

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.


Assuntos
Oligonucleotídeos/síntese química , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/química , RNA Interferente Pequeno/química , Proteínas Supressoras de Tumor/genética , Animais , Portadores de Fármacos , Estabilidade de Medicamentos , Células HeLa , Humanos , Camundongos , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , PTEN Fosfo-Hidrolase , Fosfatidiletanolaminas , Monoéster Fosfórico Hidrolases/biossíntese , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Transfecção , Proteínas Supressoras de Tumor/biossíntese
19.
J Med Chem ; 48(4): 1199-210, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15715486

RESUMO

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.


Assuntos
Monofosfato de Citidina/análogos & derivados , Monofosfato de Citidina/síntese química , Citidina/análogos & derivados , Citidina/química , Hepacivirus/efeitos dos fármacos , Organofosfatos/síntese química , Pró-Fármacos/síntese química , Animais , Células CHO , Linhagem Celular Tumoral , Aberrações Cromossômicas/induzido quimicamente , Cricetinae , Cricetulus , Monofosfato de Citidina/química , Monofosfato de Citidina/farmacologia , Hepacivirus/genética , Humanos , Organofosfatos/química , Organofosfatos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , RNA Viral/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Trítio , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
20.
Sci Rep ; 5: 18277, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26667495

RESUMO

The molecular origins of fibrosis affecting multiple tissue beds remain incompletely defined. Previously, we delineated the critical role of the control of extracellular matrix (ECM) stiffening by the mechanosensitive microRNA-130/301 family, as activated by the YAP/TAZ co-transcription factors, in promoting pulmonary hypertension (PH). We hypothesized that similar mechanisms may dictate fibrosis in other tissue beds beyond the pulmonary vasculature. Employing an in silico combination of microRNA target prediction, transcriptomic analysis of 137 human diseases and physiologic states, and advanced gene network modeling, we predicted the microRNA-130/301 family as a master regulator of fibrotic pathways across a cohort of seemingly disparate diseases and conditions. In two such diseases (pulmonary fibrosis and liver fibrosis), inhibition of microRNA-130/301 prevented the induction of ECM modification, YAP/TAZ, and downstream tissue fibrosis. Thus, mechanical forces act through a central feedback circuit between microRNA-130/301 and YAP/TAZ to sustain a common fibrotic phenotype across a network of human physiologic and pathophysiologic states. Such re-conceptualization of interconnections based on shared systems of disease and non-disease gene networks may have broad implications for future convergent diagnostic and therapeutic strategies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/genética , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Modelos Animais de Doenças , Matriz Extracelular , Fibrose , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Fosfoproteínas/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP
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