RESUMO
IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Metilação , Camundongos , Modelos Moleculares , Conformação Molecular , Conformação de Ácido Nucleico , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
We report the first syntheses of three nucleoside analogues, namely, 2',4'-diOMe-rU, 2'-OMe,4'-F-rU, and 2'-F,4'-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4'-α-position of a 4',5'-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2',4'-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4'-α-F/OMe substituents reinforce the C3'-endo ( north) conformation of 2'-OMe-rU. Furthermore, the predominant C2'-endo ( south/ east) conformation of 2'-F-araU switches to C3'-endo upon introduction of these substituents at C4'. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4'-substituents can modulate the binding affinity of the parent 2'-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics.
RESUMO
We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.
Assuntos
Inativação Gênica , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Ribonucleotídeos/genética , Termodinâmica , Uridina/química , Uridina/metabolismoRESUMO
We report the synthesis, thermal stability, and RNase H substrate activity of 2'-deoxy-2',4'-difluoroarabino-modified nucleic acids. 2'-Deoxy-2',4'-difluoroarabinouridine (2,'4'-diF-araU) was prepared in a stereoselective way in six steps from 2'-deoxy-2'-fluoroarabinouridine (2'-F-araU). NMR analysis and quantum mechanical calculations at the nucleoside level reveal that introduction of 4'-fluorine introduces a strong bias toward the North conformation, despite the presence of the 2'-ßF, which generally steers the sugar pucker toward the South/East conformation. Incorporation of the novel monomer into DNA results on a neutral to slightly stabilizing thermal effect on DNA-RNA hybrids. Insertion of 2',4'-diF-araU nucleotides in the DNA strand of a DNA-RNA hybrid decreases the rate of both human and HIV reverse transcriptase-associated RNase H-mediated cleavage of the complement RNA strand compared to that for an all-DNA strand or a DNA strand containing the corresponding 2'-F-araU nucleotide units, consistent with the notion that a 4'-fluorine in 2'-F-araU switches the preferred sugar conformation from DNA-like (South/East) to RNA-like (North).
Assuntos
Arabinose/análogos & derivados , DNA/química , DNA/síntese química , RNA/química , RNA/síntese química , Arabinose/química , Estrutura MolecularRESUMO
We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 µM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.
Assuntos
Antivirais/síntese química , Citidina/química , RNA Polimerases Dirigidas por DNA/química , Floxuridina/análogos & derivados , RNA/química , Ribonucleosídeos/síntese química , Proteínas não Estruturais Virais/química , Antivirais/química , RNA Polimerases Dirigidas por DNA/metabolismo , Floxuridina/síntese química , Floxuridina/química , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Conformação Molecular , Ribonucleosídeos/química , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/farmacologiaRESUMO
We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'âP5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.
Assuntos
Oligonucleotídeos Antissenso , Oligonucleotídeos , Oligonucleotídeos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Ácidos Fosfóricos/farmacologia , Ácidos Fosfóricos/química , Amidas/farmacologia , Amidas/químicaRESUMO
Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.
Assuntos
Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/química , Nucleosídeos/química , Nucleosídeos/síntese química , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Pirrolidinas/síntese química , Sulfonamidas/síntese química , Pareamento de Bases , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/química , Sulfonamidas/químicaRESUMO
A series of 2',3'-dideoxy-2',2'-difluoro-4'-azanucleosides of both pyrimidine and purine nucleobases were synthesized in an efficient manner starting from commercially available L-pyroglutamic acid via glycosylation of difluorinated pyrrolidine derivative 15. Several 4'-azanucleosides were prepared as a separable mixture of α- and ß-anomers. The 6-chloropurine analogue was obtained as a mixture of N(7) and N(9) regioisomers and their structures were identified based on NOESY and HMBC spectral data. Among the 4'-azanucleosides tested as HIV-1 inhibitors in primary human lymphocytes, four compounds showed modest activity and the 5-fluorouracil analogue (18d) was found to be the most active compound (EC(50)=36.9µM) in this series. None of the compounds synthesized in this study demonstrated anti-HCV activity.
Assuntos
Antivirais/química , Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Antivirais/síntese química , Antivirais/toxicidade , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Aza/toxicidade , Infecções por HIV/tratamento farmacológico , Halogenação , Hepatite C/tratamento farmacológico , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Nucleosídeos/síntese química , Nucleosídeos/toxicidadeRESUMO
Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.
Assuntos
Desoxirribose/análogos & derivados , Química Verde/métodos , Oligonucleotídeos/síntese química , Acilação , Biocatálise , Chromobacterium/enzimologia , Desoxirribose/química , Desoxirribose/metabolismo , Lipase/metabolismo , Oligonucleotídeos/metabolismo , Pseudomonas/enzimologiaRESUMO
We have developed a lipase-catalyzed parallel kinetic resolution of mixtures of ß-D/L-nucleosides. The opposite selectivity during acylation exhibited by Pseudomonas cepacia lipase (PSL-C) with ß-D- and ß-L-nucleosides furnished acylated compounds that have different R(f) values. As a consequence, isolation of both products was achieved by simple column chromatography. Computer modeling of the transition-state analogues during acylation of ß-D- and ß-L-2'-deoxycytidine with PSL-C was carried out to explain the high selectivity. PSL-C favored the 3'-O-levulination of the ß-D enantiomer, whereas the 5'-OH group was acylated in 2'-deoxy-ß-L-cytidine. In both cases, the cytosine base was placed in the alternate hydrophobic pocket of PSL's substrate-binding site, where it can form extra hydrogen bonds (in addition to the five essential catalytically relevant hydrogen bonds) that stabilize these intermediates catalyzing the selective acylation of ß-D/L-nucleosides.
Assuntos
Burkholderia cepacia/enzimologia , Lipase/metabolismo , Nucleosídeos/química , Biocatálise , Cinética , Lipase/química , Conformação de Ácido Nucleico , Nucleosídeos/isolamento & purificação , EstereoisomerismoRESUMO
In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.
Assuntos
Proteínas Argonautas/genética , Inativação Gênica , RNA Interferente Pequeno/genética , Açúcares/química , Proteínas Argonautas/antagonistas & inibidores , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla , RNA Interferente Pequeno/antagonistas & inibidores , Uridina/químicaRESUMO
Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide (AON) conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the GSCs, such as CD44 and EphA2, conjugated to chemically modified AONs against DRR/FAM107A, which were designed as chimeras of DNA and 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.
RESUMO
Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2'-deoxy-2'-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets.
RESUMO
2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the "neutral" effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2',4'-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2',4'-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2'-F-ANA modifications, and as active as the 2'-F-RNA modified siRNAs.
Assuntos
DNA/química , Floxuridina/análogos & derivados , RNA Interferente Pequeno/química , RNA/química , Sequência de Bases , Floxuridina/química , Células HeLa , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
The synthesis of a novel series of seven-membered ring nucleoside analogues as candidates for biological screening and gene silencing applications is described. The key step in the synthetic approach is a stereoselective synthesis of an epoxide that is used as a common synthetic intermediate to prepare functionalized oxepane nucleoside derivatives. The conformational landscape and preferred ring-puckering of selected oxepane nucleosides was also studied by NMR, X-ray crystallography, and quantum mechanical calculations.
Assuntos
Nucleosídeos/química , Nucleosídeos/síntese química , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , EstereoisomerismoRESUMO
A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT. We found that DNA primers that contain North puckered-nucleotides at the 3'-terminus can also affect the translocation status of the RT/template/primer complex, which provides an underlying mechanism to avoid being excised. Together, these results point to a correlation between the sugar conformation of the 3'-terminal nucleotide, the precise position of HIV-1 RT on its nucleic acid substrate, and, in turn, its catalytic function. Nucleotide sugar conformation is therefore an important parameter in defining the susceptibility to RT-catalyzed phosphorolytic excision.