In vitro anti-HIV activity of phosphorothioate alpha-anomeric oligodeoxynucleotides

Oligonucleotide analogs consisting exclusively of alpha-anomeric deoxynucleoside units bridged with phosphorothioate linkages have been synthesized and tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Two 28-mers, an homopolymer alpha-S-dC28 and an oligomer alpha-S-anti-rev complementary to the initiation site of the regulatory viral gene rev exhibited antiviral activities comparable to those reported for the corresponding beta-anomeric phosphorothioate analogs. In contrast, a nuclease-resistant homopolymer, alpha-dC28 was inactive. Their preliminary results would indicate that the origin of oligonucleotide phosphorothioate anti-HIV activity is not exclusively correlated with their higher nuclease resistance.     

Protection of oligonucleotide primers against degradation by DNA polymerase I

By use of a mutational assay employing an octadecamer with a mismatch in the center, it is shown that the introduction of phosphorothioate groups near the 5'-end can protect the mismatch against degradation by the 5'-3'-exonuclease activity of Escherichia coli DNA polymerase I. An optimal level of protection is achieved when the phosphorothioate groups are incorporated in at least the second and third internucleotidic linkages from the 5'-end. However, gel electrophoretic analysis as well as the use of an octadecamer with a mismatch closer to the 5'-end in the mutational assay reveals that degradation of the oligonucleotide is not completely blocked but only slowed down.     

2′-SUBSTITUTED PHOSPHOROTHIOATE CONTAINING OLIGORIBONUCLEOTIDES: AN APPLICATION TO THE SYNTHESIS AND PURIFICATION OF HAMMERHEAD RIBOZYMES

Abstract

A systematic study of the catalytic activity and nuclease stability of selectively modified hammerhead ribozymes has resulted in the identification of a generic motif containing 5 ribose residues and 31 2′- modified sugars (1). This substructure has been further elaborated to include phosphorothioate linkages. Although oligodeoxyribonucleotides containing phosphorothioate linkages have been studied extensively, similarly substituted RNA molecules or ribozymes have not been explored at-length. The synthesis and purification of these ribozymes is discussed (2).     

Synthesis of Anti-Sense Phosphorothioate Analogues for Pharmacokinetic and Pre-Clinical Studies

Abstract

Nudease-resistant oligonucleotides (11 to 28-mers) containing stereorandom phosphorothioate linkages have been recently reported to exhibit potent anti-HIV III effects and sequence-specific inhibition of protein synthesis. Relatively large amounts (100 mg 1g) of these analogues. which are needed for further biological testing and initial pharmacokinetic and pre-clinical studies, were readily obtained by automated hydrogen phosphonate chemistry followed by reversed-phase HPLC and further processing. This chemistry features 1 -adamanetanecarbonyl chloride as the activator, capping with isopropyl phosphite, and more complete sulfurization in only one-step following chain assembly. An automated, quantitative. picomole method for analysis of the analogues in blood samples has been developed.     

Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.     

Protein and RNA of Human Telomerase as Targets for Modified Oligonucleotides

Abstract

Chimeric oligodeoxynucleotides containing phosphorothioate and N3′→P5′phosphoramidate linkages were synthesized. These oligomers show a high inhibitory activity against human telomerase.     

Phosphorothioate analogues of oligodeoxyribonucleotide: synthesis and activity as inhibitors of replication of human immunodeficiency virus

The modifications of oligodeoxyribonucleotides include replacement of the other chain either all-PS (S-ODNs), or end-capped with several PS (SO-ODNs) groups at both 3'- and 5'-ends. A general synthesis of phosphorothioate analogues of oligodeoxyribonucleotides is described using the new phosphite. In assays of HIV, oligomers (S-ODNs) with complete replacement of phosphodiesters with phosphorothioate groups were found to be very active. Finally of particular interest is S-ODNs-rev or tat (20mers) which possessed slightly higher anti-HIV activity than S-dC28 itself. By contrast, the unmodified oligomers (ODNs) as well as SO-ODNs did not have any inhibitory effect.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Effect of structural variations in cholesteryl-conjugated oligonucleotides on inhibitory activity toward HIV-1.

A number of oligonucleotide analogues containing internucleoside phosphorothioate linkages and a covalently attached cholesteryl residue was synthesized and tested for activity against HIV-1 in cultures of Molt3 cells. Structural features important for high antiviral activity are the presence of a cholesteryl moiety, a run of terminal phosphorothioate groups, and the presence of nucleoside residues. An increase in length of the tether between cholesteryl and phosphorus from six to 14 atoms has no significant effect on antiviral activity, and up to one-half of the internucleoside links in a cholesteryl-conjugated phosphorothioate oligomer and one-third of the internucleoside links in a nonconjugated phosphorothioate can be replaced with phosphodiester links without much change in antiviral activity. However, replacement of nucleoside units in the oligomers by a simple analogue (-OCH2CH2CH2O-) yields inactive or very weakly active compounds, even in the presence of a cholesteryl group. Dose-response patterns for assays in which cholesteryl-conjugated oligomers are added to test cells either simultaneously or subsequently to viral infection are similar for homooligomer derivatives and for oligomers containing "antisense" sequences, suggesting a similarity in mode of action for the two classes of oligomers in this system.     

Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives.

Several synthetic 2'-O-methyl-RNA oligomers and their derivatives have been evaluated for inhibitory effect against HIV-induced cytopathic effect and expression of the virus specific antigen in cultured MT-4 cells. In this study, oligo(2'-O-methyl)ribonucleoside phosphorothioates showed a potent inhibitory activity with size dependency (25-mer showed it at 1 microM), but by contrast both 2'-O-methylribo- and deoxy-oligomers with normal phosphate linkages failed to inhibit. However, it should be noted that the patched oligo(2'-O-methyl)ribonucleotide (20-mer), in which five linkages at 5'- and three linkages at 3'-ends of normal phosphates were replaced with thiophosphates, has recovered the substantial inhibitory effect. These results show that the size of oligomer and phosphorothioate linkages, probably resistant to exolytic nucleases, are essential for exhibiting antiviral activity.     

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.     

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.     

Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor elF4A.

Eukaryotic translation initiation factor 4A (elF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5' untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5'-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to elF4A and on stimulation of its ATPase activity once bound. Substitution of 2'-OH groups with 2'-H or 2'-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2'-OH groups participate in an elF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to approximately 15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2'-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2'-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and elF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow elF4A to operate within regions of 5' UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5' UTR influences translation efficiency.     

Second Generation Antisense Oligonucleotides—Inhibition of PKC-α and c-raf Kinase Expression by Chimeric Oligonucleotides Incorporating 6″-Substituted Carbocyclic Nucleosides and 2″-O-Ethylene Glycol Substituted Ribonucleosides

Abstract

6′-substituted carbocyclic deoxyribonucleosides and 2′-O-ethylene glycol substituted ribonucleosides have been evaluated as building blocks for antisense oligonucleotides. Within the former class 6′-hydroxy substituted building blocks in combination with internucleoside phosphorothioate linkages have the potential to enhance antisense activity. 2′-O-ethylene glycol substituted ribonucleosides generally allow for the construction of potent antisense oligonucleotides with reduced phosphorothioate content, but differences exist in their effects on biological activity in cell culture in spite of virtually identical effects on RNA-binding affinity. Activity enhancement was most pronounced for a 2′-O-methoxyethyl substituent.     

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.     

Synthesis And Anti-HIV Property of Novel Oligo-DNA Phosphorothioate Analogs Bearing an Intercalative Moiety and/Or Polyamine Residues

Abstract

Novel oligoDNA phosphorothioate analogs (S-ODN-1 to -3) bearing an intercalative moiety at the 5′-terminus and/or polyamine at the C-5 position of certain uridine residues substituting for normal thymidine residues were synthesized and their physicochemical properties as well as the anti-HIV activities were studied. The analogs have identical base sequence which is complementary to art/tris region of human immunodeficiency virus type 1 (HIV-1). The polyamine moiety on the analogs is found to be effective not only to enhance the hybridization ability but also reduce the nonspecific cytotoxicity and strengthen the anti-HIV activity of the analogs.     

2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression.

2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA. These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H. Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin. Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture.     

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.