RESUMO
We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.
Assuntos
Ácidos Nucleicos , Ácidos Nucleicos/química , RNA/química , Fósforo , Cinética , DNA/química , Oligonucleotídeos/química , Conformação de Ácido NucleicoRESUMO
Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.
Assuntos
Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Fosforotioatos/análise , Espectrometria de Massa de Íon Secundário/métodos , Células 3T3-L1 , Acetilgalactosamina/administração & dosagem , Acetilgalactosamina/análise , Animais , Receptor de Asialoglicoproteína/análise , Césio , Células HEK293 , Células HeLa , Humanos , Rim/química , Rim/ultraestrutura , Fígado/química , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Miocárdio/química , Miocárdio/ultraestrutura , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Fosforotioatos/farmacocinética , Pseudópodes/química , Pseudópodes/ultraestrutura , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Frações Subcelulares/química , Enxofre/análise , Isótopos de Enxofre/análise , Distribuição TecidualRESUMO
The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Índice Terapêutico do Medicamento , Animais , Células HEK293 , Células HeLa , Humanos , Fígado/metabolismo , Masculino , Mesilatos/química , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/toxicidade , Fosforamidas/química , Ligação Proteica , Distribuição TecidualRESUMO
We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.
Assuntos
DNA/química , Oligonucleotídeos Antissenso/química , Animais , Glucose/análogos & derivados , Glucose/química , Células HeLa , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/toxicidade , Compostos Organofosforados/síntese química , Ribonuclease HRESUMO
The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.
Assuntos
Hidrocarbonetos/química , RNA/química , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Organofosfonatos/química , RNA/metabolismoRESUMO
Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.
Assuntos
DNA/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/genética , Ribonuclease H/genética , Animais , DNA/química , Camundongos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Ligação Proteica/genética , Ribonuclease H/químicaRESUMO
Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismoRESUMO
The activity of PS-ASOs is strongly influenced by association with both inter- and intracellular proteins. The sequence, chemical nature, and structure of the ASO can have profound influences on the interaction of PS-ASOs with specific proteins. A more thorough understanding of how these pharmacological agents interact with various proteins and how chemical modifications, sequence, and structure influence interactions with proteins is needed to inform future ASO design efforts. To better understand the chemistry of PS-ASO interactions, we have focused on human positive cofactor 4 (PC4). Although several studies have investigated the in vitro binding properties of PC4 with endogenous nucleic acids, little is known about the chemistry of interaction of PS-ASOs with this protein. Here we examine in detail the impact of ASO backbone chemistry, 2'-modifications, and buffer environment on the binding affinity of PC4. In addition, using site-directed mutagenesis, we identify those amino acids that are specifically required for ASO binding interactions, and by substitution of abasic nucleotides we identify the positions on the ASO that most strongly influence affinity for PC4. Finally, to confirm that the interactions observed in vitro are biologically relevant, we use a recently developed complementation reporter system to evaluate the kinetics and subcellular localization of the interaction of ASO and PC4 in live cells.
Assuntos
Proteínas de Ligação a DNA/química , Oligonucleotídeos Antissenso/química , Fatores de Transcrição/química , Células HEK293 , Células HeLa , Humanos , CinéticaRESUMO
A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.
Assuntos
Regiões 5' não Traduzidas , Oligonucleotídeos Antissenso/farmacologia , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Receptores de LDL/genética , Ribonuclease H/genética , Animais , Humanos , Lipoproteínas LDL/farmacocinética , Masculino , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/química , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/química , Receptores de LDL/metabolismo , Ribonuclease H/metabolismoRESUMO
A convenient method for the synthesis of several triantennary GalNAc clusters based on a nitromethanetrispropionic acid core was developed. The synthetic approach involves pentafluorophenolic ester intermediates which can be used in a one-pot, seven reaction procedure to quickly prepare a variety of triantennary GalNAc conjugated ASOs. The GalNAc clusters were conjugated to the 5'-end of an antisense oligonucleotide and evaluated for activity in primary mouse hepatocytes where they showed â¼10-fold improvement in activity.
Assuntos
Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/síntese química , Nitrocompostos/síntese química , Oligonucleotídeos Antissenso/síntese química , Propionatos/síntese química , Acetilgalactosamina/farmacologia , Animais , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Indicadores e Reagentes , Camundongos , Nitrocompostos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Propionatos/farmacologia , Receptores Depuradores Classe B/metabolismoRESUMO
Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Animais , Células Cultivadas , Proteínas Fúngicas/metabolismo , Camundongos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ribonuclease H/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Estereoisomerismo , TemperaturaRESUMO
Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. â¼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. â¼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.
Assuntos
Acetilgalactosamina/química , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacologia , Animais , Células Cultivadas , Hepatócitos/citologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5'-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5'-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5'-triantennary GalNAc cluster conjugated ASOs and helps understand the structure-activity relationship for targeting hepatocytes with oligonucleotide therapeutics.
Assuntos
Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/síntese química , Compostos Organofosforados/química , Receptores Depuradores Classe B/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Fígado/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Depuradores Classe B/metabolismo , Relação Estrutura-AtividadeRESUMO
Approaches to the synthesis of the constrained 5-methyluracil nucleoside (S)-cEt-BNA, a key "gapmer" unit in a number of biologically relevant antisense oligonucleotides, are described using 5-methyluridine as starting material. In the shorter synthesis, a nine-step linear sequence afforded a O-protected (S)-cEt-BNA consisting of a [2.2.1]dioxabicycloheptane core in 7% overall yield. A competing reaction in an intramolecular cyclization of a tosylate led to a bicyclic oxetane.
Assuntos
Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/síntese química , Éteres Cíclicos/química , Éteres Cíclicos/síntese química , Timina/química , Timina/síntese química , Uridina/análogos & derivados , Uridina/química , Hidrocarbonetos Aromáticos com Pontes/síntese química , Ciclização , Estrutura Molecular , Nucleosídeos/síntese química , Oligonucleotídeos Antissenso/síntese químicaRESUMO
The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.
Assuntos
Oligonucleotídeos Antissenso , Oligonucleotídeos Fosforotioatos , Animais , Endossomos/metabolismo , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Fosforotioatos/genética , Fósforo , Índice TerapêuticoRESUMO
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 , EstereoisomerismoRESUMO
Incorporation of an hydrophobic (phenethylamino)ethyl ether at C2â³ of N1-(HABA)-3',4'-dideoxyparomomycin led to a novel analog with an excellent antibacterial profile against a host of resistant bacteria.
Assuntos
Aminoglicosídeos/química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Desenho de Fármacos , RNA Ribossômico/química , Antibacterianos/farmacologia , Cristalização , Farmacorresistência Bacteriana , Interações Hidrofóbicas e HidrofílicasRESUMO
A series of antisense oligonucleotides (ASOs) containing either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.
Assuntos
Fígado/efeitos dos fármacos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/toxicidade , Animais , Apoptose , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas , Fígado/patologia , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos , RatosRESUMO
The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , Humanos , Fígado/efeitos dos fármacos , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Fosforotioatos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ribonuclease H/química , Ribonuclease H/genética , Índice TerapêuticoRESUMO
A series of 2"-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit microM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses.