RESUMEN
This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described.
Asunto(s)
Antineoplásicos/farmacología , Antivirales/farmacología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Interferones/farmacología , Oligorribonucleótidos Antisentido/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Virus ARN/efectos de los fármacos , Nucleótidos de Adenina/química , Nucleótidos de Adenina/metabolismo , Animales , Antivirales/química , Antivirales/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Portadoras/síntesis química , Proteínas Portadoras/química , Endorribonucleasas/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Oligorribonucleótidos Antisentido/síntesis química , Oligorribonucleótidos Antisentido/química , Virus Sincitiales Respiratorios/efectos de los fármacos , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
This unit provides protocols for the synthesis and characterization of 2-5A-antisense nucleic acids. These chimeric oligonucleotides consist of 2',5'-phosphodiester-linked oligoadenylates ligated to 3',5'-deoxyribonucleotides and are readily prepared using phosphoramidite chemistry on CPG solid supports. The 3',5'-deoxyribonucleotide functions as the antisense domain to target a given mRNA sequence, while the 2',5'-phosphodiester-linked oligoadenylate serves to locally activate 2-5A-dependent RNase L, causing the targeted sequence to be cleaved.
Asunto(s)
Nucleótidos de Adenina/química , Nucleótidos de Adenina/síntesis química , Bioquímica/métodos , ADN/química , ADN/síntesis química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/síntesis química , Oligorribonucleótidos/química , Oligorribonucleótidos/síntesis química , Animales , Cromatografía Líquida de Alta Presión , Oligonucleótidos Antisentido/aislamiento & purificación , Oligonucleótidos Antisentido/metabolismo , Compuestos Organofosforados/química , Hidrolasas Diéster Fosfóricas/metabolismo , Análisis de Secuencia de ADN , Venenos de SerpienteRESUMEN
2-5A was conjugated to N-(2-aminoethyl)-glycyl PNA by periodate oxidization, followed by coupling with amino-derivatized PNA and final cyanoborohydride reduction. An adduct of 2-5A pentamer with tetrameric thymine PNA activated RNase L with the same potency as earlier versions of 2-5A-PNA or 2-5A-DNA.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas/metabolismo , Oligorribonucleótidos/química , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismoRESUMEN
Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.
Asunto(s)
Nucleótidos de Adenina/química , ADN sin Sentido/química , Exonucleasas/metabolismo , Nucleótidos/química , Oligorribonucleótidos/química , Secuencia de Bases , Endorribonucleasas/metabolismo , Activación Enzimática , Humanos , ARN Mensajero/genéticaRESUMEN
A method is described for monitoring the cleavage of an oligoribonucleotide substrate by the 2-5A-dependent RNase L based on fluorescence resonance energy transfer (FRET). The oligoribonucleotide, rC11U2C7, was labeled covalently at its 5'-terminus with fluorescein and at its 3'-terminus with rhodamine to provide a substrate for RNase L. On cleavage, the fluorescence at 538 nm (with 485 nm excitation) increased by a factor of 2.8, allowing real-time quantitation of the reaction progress. The method was performed easily in a 96-well plate format and allowed quantitative high throughput analyses of RNase L activity with different activators.
Asunto(s)
Endorribonucleasas/química , Oligorribonucleótidos/química , Cromatografía Líquida de Alta Presión , Activación Enzimática , Fluoresceína/química , Colorantes Fluorescentes/química , Cinética , Estructura Molecular , Oligorribonucleótidos/metabolismo , Rodaminas/química , Espectrometría de FluorescenciaRESUMEN
2-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me2A), p5'(me2A)2'p5'A2'p5'A, and p5'(me2A) 2'p5'(me2A)2'pS'(me2A), were prepared via a modification of a lead ion-catalyzed ligation reaction. These 5'-monophosphates were subsequently converted into the corresponding 5'-triphosphates. Both binding and activation of human recombinant RNase L by various 2-methyladenosine-substituted 2-5A analogues were examined. Among the 2-5A analogues, p5'A2'p5'A2'p5'(me2A) showed the strongest binding affinity and was as effective as 2-5A itself as an activator of RNase L. The CD spectra of both p5'(me2A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(me2A) were superimposable on that of p5'A2'p5'A2'p5'A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2-5A.
Asunto(s)
Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Adenosina/análogos & derivados , Endorribonucleasas/efectos de los fármacos , Oligorribonucleótidos/química , Oligorribonucleótidos/farmacología , Adenosina/química , Adenosina/farmacología , Adenosina Monofosfato/análogos & derivados , Sitios de Unión , Catálisis/efectos de los fármacos , Dicroismo Circular , Endorribonucleasas/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Unión Proteica , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children, and is also a significant threat to other populations including the immunosuppressed, the elderly and those with chronic chest or cardiac disease. To expand the scope of available antiviral drugs, presently limited to ribavirin, a variety of different structural formats have been explored in the past half-dozen years. Interesting leads for future discovery and lead development include a group of biphenyl relatives (represented by CL-387626) that bind the RSV fusion (F) protein; 2-5A-antisense oligonucleotides that target the RSV genomic RNA; Rho A-derived peptides that block Rho A GTPase interaction with RSV fusion (F) protein; and several compounds of presently unknown mechanisms of action, such as benzodithiins.
Asunto(s)
Nucleótidos de Adenina/genética , Endorribonucleasas/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligorribonucleótidos/genética , eIF-2 Quinasa/genética , Nucleótidos de Adenina/química , Animales , Baculoviridae , Quimera , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Endorribonucleasas/aislamiento & purificación , Activación Enzimática , Vectores Genéticos , Humanos , Cinética , Oligodesoxirribonucleótidos Antisentido/síntesis química , Oligodesoxirribonucleótidos Antisentido/química , Oligorribonucleótidos/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera , Especificidad por Sustrato , TransfecciónRESUMEN
Fully automated solid-phase synthesis gave access to a hybrid in which 5'-phosphorylated-2'-5'-linked oligoadenylate (2-5A) is connected to the 5'-terminus of DNA which, in turn, is linked at the 3'-end to PNA [2-5A-(5')-DNA-(3')-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.
Asunto(s)
Endorribonucleasas/síntesis química , Oligonucleótidos Antisentido/metabolismo , Ácidos Nucleicos de Péptidos , Cromatografía Líquida de Alta Presión , Endorribonucleasas/metabolismo , Activación Enzimática , Humanos , Unión ProteicaRESUMEN
The development of a method for measuring the ability of 2-5A analogues to activate the cleavage of an oligoribonucleotide substrate by RNase L is described. This method is based on fluorescence resonance energy transfer. The method is easily performed with 96-well plates, allowing for quantitative high-throughput analyses of 2-5A analogues under different reaction conditions.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas , Oligorribonucleótidos/química , Nucleótidos de Adenina/farmacología , Transferencia de Energía , Activación Enzimática , Humanos , Cinética , Oligonucleótidos Antisentido/química , Oligorribonucleótidos/farmacología , Espectrometría de FluorescenciaRESUMEN
This paper presents the fully automated solid phase synthesis of 2-5A-PNA hybrids. These stable antisense probes cause RNase L mediated hydrolysis of target RNA sequences.
Asunto(s)
Oligonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/química , Secuencia de Bases , Endorribonucleasas/química , HidrólisisRESUMEN
2',5'-Oligoadenylate (2-5A) antisense chimeric oligonucleotides were synthesized containing varying 2'-O-methyl-ribonucleotide substitution patterns in the antisense domain. The ability of these composite oligonucleotides to mediate RNase H- and RNase L-catalyzed RNA degradation showed that these two enzymes have different activation requirements.
Asunto(s)
Nucleótidos de Adenina/genética , Endorribonucleasas/metabolismo , Oligonucleótidos Antisentido/química , Oligorribonucleótidos/genética , ARN/metabolismo , Ribonucleasa H/metabolismo , Secuencia de Bases , Hidrólisis , MetilaciónRESUMEN
To define more fully the conditions for 2-5A-antisense inhibition of respiratory syncytial virus (RSV), relationships between 2-5A antisense oligonucleotide structure and the choice of RNA target sites to inhibition of RSV replication have been explored. The lead 2-5A-antisense chimera for this study was the previously reported NIH8281 that targets the RSV M2 RNA. We have confirmed and extended the earlier study by showing that NIH8281 inhibited RSV strain A2 replication in a variety of antiviral assays, including virus yield reduction assays performed in monkey (EC90 = 0.02 microM) and human cells (EC90 = microM). This 2-5A-antisense chimera also inhibited other A strains, B strains and bovine RSV in cytopathic effect inhibition and Neutral Red Assays (EC50 values = 0.1-1.6 microM). The 2'-O-methylation modification of NIH8281 to increase affinity for the complementary RNA and provide nuclease resistance, the introduction of phosphothioate groups in the antisense backbone to enhance resistance to exo- and endonucleases, and the addition of cholesterol to the 3'-terminus of the antisense oligonucleotide to increase cellular uptake, all resulted in loss of activity. Of the antisense chimeras targeting other RSV mRNAs (NS1, NS2, P, M. G, F, and L), only those complementary to L mRNA were inhibitory. These results suggest that lower abundance mRNAs may be the best targets for 2-5A-antisense; moreover, the active 2-5A antisense chimeras in this study may serve as useful guides for the development of compounds with improved stability, uptake and anti-RSV activity.
Asunto(s)
Nucleótidos de Adenina/farmacología , Endorribonucleasas/metabolismo , Oligonucleótidos Antisentido/farmacología , Oligorribonucleótidos/farmacología , ARN Viral/metabolismo , Virus Sincitiales Respiratorios/efectos de los fármacos , Nucleótidos de Adenina/química , Nucleótidos de Adenina/genética , Animales , Secuencia de Bases , Bovinos , Línea Celular , Humanos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/química , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , ARN Mensajero/metabolismo , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/fisiología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
To potentiate the 2-5A (2',5'-oligoadenylate)-antisense and peptide nucleic acid (PNA) approaches to regulation of gene expression, composite molecules were generated containing both 2-5A and PNA moieties. 2-5A-PNA adducts were synthesized using solid-phase techniques. Highly cross-linked polystyrene beads were functionalized with glycine tethered through a p-hydroxymethylbenzoic acid linker and the PNA domain of the chimeric oligonucleotide analogue was added by sequential elongation of the amino terminus with the monomethoxytrityl protected N-(2-aminoethyl)-N-(adenin-1-ylacetyl)glycinate. Transition to the 2-5A domain was accomplished by coupling of the PNA chain to dimethoxytrityl protected N-(2-hydroxyethyl)-N-(adenin-1-ylacetyl)glycinate. Finally, (2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4-dimethoxytrityl)butylphosphor amidite and the corresponding (2-cyanoethyl)-N,N-diisopropylphosphoramidite of 5-O-(4,4'-dimethoxytrityl)-3-O-(tert-butyldimethylsilyl)-N6-benzoyladeno sine were the synthons employed to add the 2 butanediol phosphate linkers and the four 2',5'-linked riboadenylates. The 5'-phosphate moiety was introduced with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2-cyanoethyl) -N,N-diisopropylphosphoramidite. Deprotection with methanolic NH3 and tetraethylammonium fluoride afforded the desired products, 2-SA-pnaA4, 2-5A-pnaA8 and 2-5A-pnaA12. When evaluated for their ability to cause the degradation of two different RNA substrates by the 2-5A-dependent RNase L, these new 2-5A-PNA conjugates were found to be potent RNase L activators. The union of 2-5A and PNA presents fresh opportunities to explore the biological and therapeutic implications of these unique approaches to antisense.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas/metabolismo , Oligorribonucleótidos/química , Ácidos Nucleicos de Péptidos/farmacología , Línea Celular , Activación Enzimática , Humanos , Hidrólisis , Ácidos Nucleicos de Péptidos/química , Ensayo de Unión Radioligante , Proteínas Recombinantes/metabolismoRESUMEN
Phosphorothioate oligodeoxyribonucleotides were found to be inhibitors of the 2-5A-dependent RNase L. Inhibitory potency depended upon the chain length of the phosphorothioate oligonucleotide and was dependent on the phosphorothioate substitution pattern, but was not substantially base-dependent.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Tionucleótidos/farmacología , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/químicaRESUMEN
A new approach to the chemical control of respiratory syncytial virus infection is reviewed in the context of the biology of the virus and previous treatment approaches. Conjugation of the interferon action mediator, 2',5'-oligoadenylate (2-5A), to antisense agents provides a strategy for the selective ablation of RNA. This application of this technology to respiratory syncytial virus has provided a potent selective inhibitor of virus replication.
Asunto(s)
Nucleótidos de Adenina/farmacología , Elementos sin Sentido (Genética)/farmacología , Antivirales/farmacología , Interferones/fisiología , Oligorribonucleótidos/farmacología , ARN Bicatenario/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/química , Adenosina Trifosfato/metabolismo , Elementos sin Sentido (Genética)/química , Niño , Preescolar , Simulación por Computador , Diseño de Fármacos , Endorribonucleasas/metabolismo , Inducción Enzimática , Humanos , Lactante , Modelos Moleculares , Oligorribonucleótidos/química , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/fisiología , Especificidad por Sustrato , Replicación Viral/efectos de los fármacosRESUMEN
We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.
Asunto(s)
Endorribonucleasas/genética , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , ARN Mensajero/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Nucleótidos de Adenina/genética , Nucleótidos de Adenina/farmacología , Nucleótidos de Adenina/uso terapéutico , Endorribonucleasas/farmacología , Activación Enzimática/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Oligorribonucleótidos/genética , Oligorribonucleótidos/farmacología , Oligorribonucleótidos/uso terapéutico , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Células Tumorales CultivadasRESUMEN
RNase L is a latent endonuclease found in reptiles, birds, and mammals. It is activated by the 2',5'-phosphodiester-linked oligoadenylates called 2-5A and has been implicated in the mechanism of action of interferon, as well as in a variety of other biological phenomena such as apoptosis. Covalent linkage of 2-5A to antisense oligonucleotides permits recruitment of RNase L for enhancement of antisense action. The purification of RNase L described herein and the assays for its detection and activation will help to provide further mechanistic details on how this unique nuclease functions and what its biochemical roles may be. In addition, such assays will facilitate the screening of 2-5A-antisense congeners for exploration of the potential therapeutic applications of RNase L.
Asunto(s)
Endorribonucleasas/aislamiento & purificación , Animales , Western Blotting , Catálisis , Cromatografía Liquida/métodos , Endorribonucleasas/análisis , Endorribonucleasas/metabolismo , Activación Enzimática , Ratones , Etiquetas de Fotoafinidad , Poli U/metabolismo , Pruebas de Precipitina , Unión Proteica , ARN Ribosómico/metabolismo , Especificidad por SustratoRESUMEN
The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system's RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication.