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1.
Invest New Drugs ; 38(3): 844-854, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31385109

RESUMO

Background Endothelin B receptor (ETBR) is involved in melanoma pathogenesis and is overexpressed in metastatic melanoma. The antibody-drug conjugate DEDN6526A targets ETBR and is comprised of the humanized anti-ETBR monoclonal antibody conjugated to the anti-mitotic agent monomethyl auristatin E (MMAE). Methods This Phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity of DEDN6526A (0.3-2.8 mg/kg) given every 3 weeks (q3w) in patients with metastatic or unresectable cutaneous, mucosal, or uveal melanoma. Results Fifty-three patients received a median of 6 doses of DEDN6526A (range 1-49). The most common drug-related adverse events (>25% across dose levels) were fatigue, peripheral neuropathy, nausea, diarrhea, alopecia, and chills. Three patients in dose-escalation experienced a dose-limiting toxicity (infusion-related reaction, increased ALT/AST, and drug-induced liver injury). Based on cumulative safety data across all dose levels, the recommended Phase II dose (RP2D) for DEDN6526A was 2.4 mg/kg intravenous (IV) q3w. The pharmacokinetics of antibody-conjugated MMAE and total antibody were dose-proportional at doses ranging from 1.8-2.8 mg/kg. A trend toward faster clearance was observed at doses of 0.3-1.2 mg/kg. There were 6 partial responses (11%) in patients with metastatic cutaneous or mucosal melanoma, and 17 patients (32%) had prolonged stable disease ≥6 months. Responses were independent of BRAF mutation status but did correlate with ETBR expression. Conclusion DEDN6526A administered at the RP2D of 2.4 mg/kg q3w had an acceptable safety profile and showed evidence of anti-tumor activity in patients with cutaneous, mucosal, and uveal melanoma. ClinicalTrials.gov identifier: NCT01522664.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antagonistas do Receptor de Endotelina B/uso terapêutico , Imunoconjugados/uso terapêutico , Melanoma/tratamento farmacológico , Receptor de Endotelina B/metabolismo , Neoplasias Uveais/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Esquema de Medicação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
RNA ; 18(10): 1796-804, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22912485

RESUMO

miRNAs are ∼22-nt RNAs that bind to the Argonaute family of proteins and have important regulatory roles in plants and animals. Here, we show that miRNAs exhibit targeting activity in cells when delivered as single strands that are 5'-phosphorylated and that contain 2'-fluoro ribose modifications. Length preferences, chemical modification sensitivity, and genome-wide seed-based targeting all suggest that this activity is Ago-based. Activity could be enhanced by annealing of segmented passenger strands containing non-nucleic acid spacers. Furthermore, screening of randomly generated sequences identified pyrimidine rich 3' cassette sequences that increased single strand activity. These results provide an initial step in the development of single-stranded miRNA mimics for therapeutic use.


Assuntos
DNA de Cadeia Simples/síntese química , MicroRNAs/química , Mimetismo Molecular , Sequência de Bases , Clonagem Molecular , DNA de Cadeia Simples/química , Fluoretos/síntese química , Fluoretos/química , Técnicas de Silenciamento de Genes/instrumentação , Técnicas de Silenciamento de Genes/métodos , Células HCT116 , Ensaios de Triagem em Larga Escala , Humanos , Análise em Microsséries , Mimetismo Molecular/fisiologia , Dados de Sequência Molecular , Fosforilação , Ribose/síntese química , Ribose/química , Transfecção
3.
Bioconjug Chem ; 25(12): 2222-32, 2014 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-25398098

RESUMO

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Relação Estrutura-Atividade , Alcinos/química , Azidas/química , Catálise , Cromatografia Líquida de Alta Pressão/métodos , Química Click , Cobre/química , Reação de Cicloadição , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Técnicas de Síntese em Fase Sólida
4.
Nucleic Acids Res ; 40(9): 4125-36, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22253019

RESUMO

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.


Assuntos
Interferência de RNA , Pequeno RNA não Traduzido/química , Animais , Proteínas Argonautas/metabolismo , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , Pequeno RNA não Traduzido/metabolismo
5.
J Lipid Res ; 53(5): 859-867, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22355095

RESUMO

Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diacilglicerol O-Aciltransferase/deficiência , Diacilglicerol O-Aciltransferase/genética , Fígado Gorduroso/genética , Inativação Gênica , RNA Interferente Pequeno/genética , Animais , Apolipoproteínas B/deficiência , Apolipoproteínas B/genética , Colesterol/sangue , Fígado Gorduroso/sangue , Fígado Gorduroso/enzimologia , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Triglicerídeos/metabolismo
6.
RNA ; 16(12): 2336-40, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20971811

RESUMO

microRNAs are ∼ 22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. Here, we show that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3' half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3' half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics.


Assuntos
Marcação de Genes/métodos , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Deleção de Sequência/fisiologia , Sequência de Bases/genética , Sequência de Bases/fisiologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/fisiologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ribonuclease III/metabolismo , Ribonuclease III/fisiologia , Deleção de Sequência/genética , Moldes Genéticos , Transfecção , Pequeno RNA não Traduzido
7.
Nucleic Acids Res ; 38(2): 660-71, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19917641

RESUMO

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.


Assuntos
Nucleosídeos/química , Interferência de RNA , RNA Interferente Pequeno/química , Animais , Apolipoproteínas B/genética , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Fosforilação , Fosfotransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Termodinâmica , Fatores de Transcrição/metabolismo , Pequeno RNA não Traduzido
8.
J Lipid Res ; 52(4): 679-87, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21262787

RESUMO

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.


Assuntos
Anticolesterolemiantes/uso terapêutico , Azetidinas/uso terapêutico , Fluorbenzenos/uso terapêutico , Pirimidinas/uso terapêutico , Serina Endopeptidases/metabolismo , Sulfonamidas/uso terapêutico , Animais , Apolipoproteínas B/sangue , Colesterol/sangue , LDL-Colesterol/sangue , Ensaio de Imunoadsorção Enzimática , Ezetimiba , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/terapia , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosuvastatina Cálcica , Serina Endopeptidases/genética , Triglicerídeos/sangue
9.
Mol Pharmacol ; 79(6): 953-63, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21427169

RESUMO

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjögren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.


Assuntos
Inativação Gênica , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase
10.
J Am Chem Soc ; 133(24): 9200-3, 2011 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-21612237

RESUMO

Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2'-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory 'hot spots' within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation.


Assuntos
Materiais Biomiméticos/efeitos adversos , Materiais Biomiméticos/química , Sistema Imunitário/efeitos dos fármacos , MicroRNAs/genética , RNA de Cadeia Dupla/efeitos adversos , RNA de Cadeia Dupla/química , Ribose/química , Animais , Sequência de Bases , Materiais Biomiméticos/síntese química , Linhagem Celular Tumoral , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Sistema Imunitário/metabolismo , Camundongos , RNA de Cadeia Dupla/síntese química , RNA de Cadeia Dupla/genética
11.
J Am Chem Soc ; 133(42): 16766-9, 2011 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-21942264

RESUMO

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.


Assuntos
Nucleosídeos/química , Interferência de RNA , Sequência de Bases , Dados de Sequência Molecular , Estrutura Molecular , Nucleosídeos/genética
12.
Mol Cancer Ther ; 8(4): 930-9, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19372566

RESUMO

Aurora kinases play key roles in regulating centrosome maturation, mitotic spindle formation, and cytokinesis during cell division, and are considered promising drug targets due to their frequent overexpression in a variety of human cancers. SNS-314 is a selective and potent pan Aurora inhibitor currently in a dose escalation phase 1 clinical trial for the treatment of patients with advanced solid tumors. Here, we report the antiproliferative effects of SNS-314 in combination with common chemotherapeutics in cell culture and xenograft models. The HCT116 colorectal carcinoma cell line, with intact or depleted p53 protein levels, was treated with SNS-314 and a cytotoxic chemotherapeutic from a panel comprised of gemcitabine, 5-fluorouracil (5-FU), carboplatin, daunomycin, SN-38 (the active metabolite of irinotecan), docetaxel, and vincristine. Combinations were administered under either concurrent or sequential schedules. SNS-314 has predominantly additive effects when administered concurrently with commonly used anticancer agents. Sequential administration of SNS-314 with chemotherapeutic compounds showed additive antiproliferative effects with carboplatin, gemcitabine, 5-FU, daunomycin, and SN-38, and synergy was observed in combination with gemcitabine, docetaxel, or vincristine. The most profound antiproliferative effects were observed with sequential administration of SNS-314 followed by docetaxel or vincristine. In vivo, SNS-314 potentiated the antitumor activity of docetaxel in xenografts. Both the in vitro synergies observed between SNS-314 and agents that target the mitotic spindle and the potentiation seen with docetaxel in vivo are consistent with a mechanism of action in which Aurora inhibition bypasses the mitotic spindle assembly checkpoint and prevents cytokinesis, augmenting subsequent spindle toxin-mediated mitotic catastrophe and cell death.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Microtúbulos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Aurora Quinases , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Fuso Acromático/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Bioorg Med Chem Lett ; 19(17): 5158-61, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19646866

RESUMO

This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.


Assuntos
Antineoplásicos/química , Benzimidazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Aurora Quinases , Benzimidazóis/síntese química , Benzimidazóis/farmacocinética , Linhagem Celular Tumoral , Humanos , Camundongos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade
14.
Mol Ther Nucleic Acids ; 16: 367-377, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-30991218

RESUMO

Drug combinations can improve the control of diseases involving redundant and highly regulated pathways. Validating a multi-target therapy early in drug development remains difficult. Small interfering RNAs (siRNAs) are routinely used to selectively silence a target of interest. Owing to the ease of design and synthesis, siRNAs hold promise for combination therapies. Combining siRNAs against multiple targets remains an attractive approach to interrogating highly regulated pathways. Currently, questions remain regarding how broadly such an approach can be applied, since siRNAs have been shown to compete with one another for binding to Argonaute2 (Ago2), the protein responsible for initiating siRNA-mediated mRNA degradation. Mathematical modeling, coupled with in vitro and in vivo experiments, led us to conclude that endosomal escape kinetics had the highest impact on Ago2 depletion by competing lipid-nanoparticle (LNP)-formulated siRNAs. This, in turn, affected the level of competition observed between them. A future application of this model would be to optimize delivery of desired siRNA combinations in vitro to attenuate competition and maximize the combined therapeutic effect.

15.
Nat Biotechnol ; 21(3): 308-14, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12563278

RESUMO

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.


Assuntos
Inibidores de Caspase , Caspases/química , Técnicas de Química Combinatória/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Células Jurkat/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/classificação , Humanos , Células Jurkat/metabolismo , Espectrometria de Massas/métodos , Modelos Moleculares , Biblioteca de Peptídeos
16.
Nucleic Acid Ther ; 22(3): 205-10, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22519815

RESUMO

The immune stimulation induced by short interfering RNAs (siRNAs) has been reported to be quieted or abrogated by methoxy or fluoro modifications of the 2' position of the ribose sugar. However, variables such as the type of modification, nucleotide preference, and strand bias have not been systematically evaluated. Here, we report the results of a screen of several modified siRNAs via a human peripheral blood monocyte cytokine induction assay. Unlike corresponding modifications of guanosine, cytidine, or uridine, 2'-fluoro modification of adenosine significantly reduced cytokine induction while retaining siRNA knockdown activity. The results of this study suggest adenosine as an optimal target for modification.


Assuntos
Adenosina/química , RNA Interferente Pequeno/imunologia , Citocinas/biossíntese , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , RNA Interferente Pequeno/química , beta-Galactosidase/metabolismo
17.
Nucleic Acid Ther ; 22(2): 90-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22400991

RESUMO

Endogenous and exogenous short interfering RNAs (siRNAs) require a 5'-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5'-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5'-phosphate compared to their unphosphorylated versions. siRNAs that contained a 5'-phosphate had the same activity in vivo compared with unphosphorylated siRNAs, indicating phosphorylation of an siRNA is not a rate limiting step in vivo.


Assuntos
Fosfatos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Apolipoproteínas B/biossíntese , Apolipoproteínas B/genética , Sequência de Bases , Linhagem Celular , Genes Reporter , Prolina Dioxigenases do Fator Induzível por Hipóxia , Luciferases/biossíntese , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/genética , Regiões Promotoras Genéticas , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Mol Ther Nucleic Acids ; 1: e5, 2012 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-23344622

RESUMO

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

19.
Cancer Chemother Pharmacol ; 65(4): 707-17, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19649632

RESUMO

PURPOSE: The Aurora family of serine/threonine kinases (Aurora-A, Aurora-B, and Aurora-C) plays a key role in cells orderly progression through mitosis. Elevated expression levels of Aurora kinases have been detected in a high percentage of melanoma, colon, breast, ovarian, gastric, and pancreatic tumors. We characterized the biological and pharmacological properties of SNS-314, an ATP-competitive, selective, and potent inhibitor of Aurora kinases. METHODS: We studied the biochemical potency and selectivity of SNS-314 to inhibit Aurora kinases A, B, and C. The inhibition of cellular proliferation induced by SNS-314 was evaluated in a broad range of tumor cell lines and correlated to inhibition of histone H3 phosphorylation, inhibition of cell-cycle progression, increase in nuclear content and cell size, loss of viability, and induction of apoptosis. The dose and administration schedule of SNS-314 was optimized for in vivo efficacy in mouse xenograft models of human cancer. RESULTS: In the HCT116 human colon cancer xenograft model, administration of 50 and 100 mg/kg SNS-314 led to dose-dependent inhibition of histone H3 phosphorylation for at least 10 h, indicating effective Aurora-B inhibition in vivo. HCT116 tumors from animals treated with SNS-314 showed potent and sustained responses including reduction of phosphorylated histone H3 levels, increased caspase-3 and appearance of increased nuclear size. The compound showed significant tumor growth inhibition in a dose-dependent manner under a variety of dosing schedules including weekly, bi-weekly, and 5 days on/9 days off. CONCLUSIONS: SNS-314 is a potent small-molecule inhibitor of Aurora kinases developed as a novel anti-cancer therapeutic agent for the treatment of diverse human malignancies.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/prevenção & controle , Compostos de Fenilureia/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Aurora Quinase A , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Células HCT116 , Células HT29 , Células HeLa , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Compostos de Fenilureia/química , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Tiazóis/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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