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1.
Nucleic Acids Res ; 49(11): 6100-6113, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107015

RESUMO

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Pulmão/metabolismo , Morfolinos/administração & dosagem , Splicing de RNA , Animais , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Camundongos , Mutação , Peptídeos , Mucosa Respiratória/metabolismo , Transfecção
2.
Nucleic Acids Res ; 46(4): 1601-1613, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29361039

RESUMO

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure-activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.


Assuntos
Oligonucleotídeos/metabolismo , Pirazinas/farmacologia , Piridinas/farmacologia , Células HeLa , Humanos , Membranas Intracelulares/efeitos dos fármacos , Oligonucleotídeos/análise , Pirazinas/química , Piridinas/química , Relação Estrutura-Atividade
3.
Molecules ; 24(3)2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30736307

RESUMO

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1⁻oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.


Assuntos
Oligonucleotídeos/síntese química , Oligonucleotídeos/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Sintética , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Estrutura Molecular , Oligonucleotídeos/química , Splicing de RNA/efeitos dos fármacos , Relação Estrutura-Atividade
4.
Nucleic Acids Res ; 44(14): 6518-48, 2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-27084936

RESUMO

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.


Assuntos
Técnicas de Transferência de Genes , Oligonucleotídeos/uso terapêutico , Animais , Humanos , Ligantes , Ácidos Nucleicos/uso terapêutico , Receptores de Superfície Celular/metabolismo
5.
J Am Chem Soc ; 137(10): 3656-62, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25734836

RESUMO

The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF → ON and ON → OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.


Assuntos
Processamento Alternativo/efeitos da radiação , Luz , Oligonucleotídeos/genética , Animais , Células HeLa , Humanos , Oligonucleotídeos/química , Peixe-Zebra
6.
Chembiochem ; 16(9): 1284-7, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25953428

RESUMO

The construction of nanomaterials from oligonucleotides by modular assembly invariably requires the use of branched nucleic acid architectures such as three- and four-way junctions (3WJ and 4WJ). We describe the stabilization of DNA 3WJ by using non-nucleotide lipophilic spacers to create a hydrophobic pocket within the junction space. Stabilization of nucleic acid junctions is of particular importance when constructing nanostructures in the "ultra-nano" size range (<20 nm) with shorter double-stranded regions. UV thermal melting studies show that lipophilic spacers strategically placed within the junction space significantly increased thermal stability. For a 3WJ with eight base pair arms, thermal stability was increased from 30.5 °C for the unmodified junction to a maximum stability of 55.0 °C. The stability of the junction can be modulated within this temperature range by using the appropriate combinations of spacers.


Assuntos
DNA/química , Lipídeos/química , Nanoestruturas/química , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , Temperatura
7.
Nucleic Acids Res ; 41(6): 3673-87, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23396438

RESUMO

The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.


Assuntos
Benzodiazepinonas/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Animais , Benzodiazepinonas/química , Linhagem Celular , Interações Medicamentosas , Humanos , Camundongos , Camundongos SCID , Oligonucleotídeos/análise , Splicing de RNA/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia
8.
Cells ; 12(5)2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36899837

RESUMO

Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD.


Assuntos
Distrofina , Oligonucleotídeos , Animais , Camundongos , Distrofina/genética , Camundongos Endogâmicos mdx , Precursores de RNA , Oligonucleotídeos Antissenso/uso terapêutico , DNA , Éxons
9.
Bioconjug Chem ; 23(2): 147-57, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21992697

RESUMO

Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless, our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as "free" molecules or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review, we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic.


Assuntos
Endocitose , Espaço Intracelular/metabolismo , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Humanos , Oligonucleotídeos/uso terapêutico , RNA Interferente Pequeno/uso terapêutico
10.
Nucleic Acids Res ; 38(19): 6567-76, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20551131

RESUMO

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K(m) value for saturable uptake was similar to the EC(50) value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.


Assuntos
Endocitose , Oligonucleotídeos Antissenso/metabolismo , Receptores da Bombesina/metabolismo , Linhagem Celular Tumoral , Humanos , Cinética , Luciferases de Vaga-Lume/análise , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química
11.
Bioconjug Chem ; 22(11): 2210-6, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21985221

RESUMO

A series of diwalled and tetrawalled molecular umbrellas have been synthesized using cholic acid, spermidine, and lysine as starting materials. Coupling of these molecular umbrellas to an octaarginine peptide afforded agents that were capable of promoting the transport of small interfering RNA to HeLa cells, as judged by the knockdown of enhanced green fluorescent protein expression. The efficiency of this knockdown was found to increase with an increasing number of facially amphiphilic walls present, and also when a cleavable disulfide linker was replaced with a noncleavable, maleimido moiety; i.e., a group that is not susceptible to thiolate-disulfide interchange. The knockdown efficiency that was observed for one tetrawalled molecular umbrella-octaargine conjugate was comparable to that observed with a commercially available transfection agent, Lipofectamine 2000, but the conjugate showed less cytotoxicity.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Portadores de Fármacos/síntese química , Células HeLa , Humanos , Estrutura Molecular , Peptídeos/síntese química , RNA Interferente Pequeno/química
12.
Biomedicines ; 9(5)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063104

RESUMO

Antisense oligonucleotides (ASOs), siRNA and splice switching oligonucleotides (SSOs) all have immense potential as therapeutic agents, potential that is now being validated as oligonucleotides enter the clinic. However, progress in oligonucleotide-based therapeutics has been limited by the difficulty in delivering these complex molecules to their sites of action in the cytosol or nucleus of cells within specific tissues. There are two aspects to the delivery problem. The first is that most types of oligonucleotides have poor uptake into non-hepatic tissues. The second is that much of the oligonucleotide that is taken up by cells is entrapped in endosomes where it is pharmacologically inert. It has become increasingly recognized that endosomal trapping is a key constraint on oligonucleotide therapeutics. Thus, many approaches have been devised to address this problem, primarily ones based on various nanoparticle technologies. However, recently an alternative approach has emerged that employs small molecules to manipulate intracellular trafficking processes so as to enhance oligonucleotide actions. This review presents the current status of this chemical biology approach to oligonucleotide delivery and seeks to point out possible paths for future development.

13.
J Am Chem Soc ; 132(26): 8848-9, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20550198

RESUMO

Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus, there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multivalent conjugates of oligonucleotides with small-molecule ligands. Using the sigma-receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid-phase DNA synthesis. We generated mono- and trivalent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the monovalent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.


Assuntos
Espaço Intracelular/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Amidas/química , Sequência de Bases , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Ligantes , Oligonucleotídeos Antissenso/genética , Receptores sigma/metabolismo
14.
NAR Cancer ; 2(3): zcaa025, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33015625

RESUMO

Signal transduction pathways play key roles in the initiation, progression and dissemination of cancer. Thus, signaling molecules are attractive targets for cancer therapeutics and enormous efforts have gone into the development of small molecule inhibitors of these pathways. However, regrettably, there has been only moderate progress to date, primarily in connection with the RAS signaling pathway. Oligonucleotide-based drugs potentially offer several advantages for addressing signaling pathways, including their exquisite selectivity and their ability to exploit both enzymatic and nonenzymatic targets. Nonetheless, there are problems inherent in the oligonucleotide approach, not the least being the challenge of effectively delivering these complex molecules to intracellular sites within tumors. This survey article will provide a selective review of recent studies where oligonucleotides were used to address cancer signaling and will discuss both positive aspects and limitations of those studies. This will be set in the context of an overview of various cancer signaling pathways and small molecule approaches to regulate those pathways. The survey will also evaluate the challenges and opportunities implicit in the oligonucleotide-based approach to cancer signaling and will point out several possibilities for future research.

15.
ACS Chem Biol ; 15(9): 2355-2363, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786263

RESUMO

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.


Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Corantes Fluorescentes , Galectina 3/metabolismo , Galectinas/metabolismo , HIV/química , Humanos , Camundongos , Rodaminas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
16.
Nucleic Acids Res ; 35(4): 1064-74, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17264131

RESUMO

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Interferência de RNA , RNA Interferente Pequeno/química , Álcoois Açúcares/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Relação Dose-Resposta a Droga , Camundongos , Células NIH 3T3 , Fosforilação , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Rodamina 123/análise
17.
ACS Chem Biol ; 14(12): 2641-2651, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31633910

RESUMO

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Substâncias Macromoleculares/metabolismo , Citosol/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Humanos , Pirazinas/farmacologia , Piridinas/farmacologia
18.
Mol Carcinog ; 47(5): 326-37, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17932950

RESUMO

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multi-domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full-length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-delta1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Proteínas Supressoras de Tumor/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Colágeno/metabolismo , Primers do DNA , Combinação de Medicamentos , Proteínas Ativadoras de GTPase , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Laminina/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Fosfolipase C delta/metabolismo , Reação em Cadeia da Polimerase , Proteoglicanas/metabolismo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/genética , Proteína de Ligação a GTP rhoC
19.
Nucleosides Nucleotides Nucleic Acids ; 26(10-12): 1509-12, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18066816

RESUMO

A simple and efficient method for the preparation of pyrimidine 2'-O-hydroxyethoxymethylribonucleosides and 2'-O-hydroxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2' -O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells.


Assuntos
Oligorribonucleotídeos/química , Pirimidinas/química , RNA/química , Ribonucleosídeos/síntese química , Ribonucleosídeos/química
20.
ACS Chem Biol ; 12(8): 1999-2007, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28703575

RESUMO

The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.


Assuntos
Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Camundongos , Microscopia Confocal , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/química , Splicing de RNA
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