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PubMed1995

Radiolabeling of methylphosphonate and phosphorothioate oligonucleotides and evaluation of their transport in everted rat jejunum sacs.

PURPOSE: The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work. METHODS: We have developed a simple procedure for radiolabeling oligonucleotides by reductive alkylation with 14C-formaldehyde. We have utilized this approach, as well as 5' addition of fluorophores, to prepare labeled methylphosphonate and phosphorothioate oligonucleotides for use in intestinal transport studies. An everted rat gut sac model was employed to compare the transport of oligonucleotides to that of model compounds whose permeation properties are better understood. RESULTS: We demonstrate that both methylphosphonate and phosphorothioate oligonucleotides are passively transported across the intestinal epithelium, probably by a paracellular route. The rates of transport for both types of oligonucleotides were similar, and were significantly greater than that of the very high MW polymer blue dextran, but were lower than the transport rate of valproic acid, a low MW compound known to have high oral availability. CONCLUSIONS: A significant degree of permeation of oligonucleotides across the gastrointestinal epithelium does occur, but it is still unclear whether this is sufficient to permit effective oral administration of oligonucleotides as drugs.

Alkylation↗
PubMed2004

The arthritogenic and immunostimulatory properties of phosphorothioate oligodeoxynucleotides rely on synergy between the activities of the nuclease-resistant backbone and CpG motifs.

Experiments with immunostimulatory unmethylated CpG-containing DNA are usually conducted with nuclease-protected phosphorothioate oligodeoxynucleotides (S-ODNs), rather than phosphodiester oligodeoxynucleotides (O-ODNs). We compared the murine immune responses to S-ODNs and O-ODNs that either contained or lacked CpG motifs. Both CpG and non-CpG S-ODNs induced synovitis, as did sequence-matched CpG O-ODN, but not GpC O-ODN. There was a minimum length requirement for arthritogenic S-ODNs since a CpC dinucleotide S-ODN did not induce arthritis. There were both sequence- (CpG > non-CpG) and backbone-dependent (S-ODN > O-ODN) differences in the levels of DNA-induced arthritis upon intra-articular injection with the ODNs. However, CpG O-ODN being an exception, induced more severe arthritis than the GpC S-ODN. The levels of in vitro proliferation and production of IL-6, TNF-alpha, IL-12, and RANTES by splenocytes following exposure to CpG S-ODN were significantly higher than those induced by CpG O-ODN. In addition, both proliferative responses and cytokine production induced by S-ODN-stimulated splenocytes increased significantly when the S-ODN contained a CpG motif. Transcription factor NFkappaB was activated by both CpG S-ODN and CpG O-ODN but interestingly not by GpC S-ODN. This indicates that the NFkappaB signal pathway modulates CpG-mediated immunostimulation, while sequence-independent immune activation by the phosphorothioate backbone is probably signalled via a different pathway.

Adjuvants, Immunologic↗
PubMed1996

Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase.

Substantial evidence exists supporting a direct role for raf kinases in the development and maintenance of certain human malignancies. Here we test the potential of phosphorothioate antisense oligodeoxynucleotides targeted against human C-raf-1 kinase to specifically inhibit C-raf-1 kinase gene expression and tumor progression in cell culture and in vivo, using human tumor xenograft mouse models. Treatment of human tumor cells with appropriate phosphorothioate antisense oligodeoxynucleotides led to specific inhibition of C-raf kinase gene expression in cell culture and in vivo at well-tolerated doses. Moreover, oligodeoxynucleotide treatment resulted in potent antiproliferative effects in cell culture and potent antitumor effects in vivo against a variety of tumor types that were highly consistent with an antisense mechanism of action for these compounds. These studies strongly suggest that antisense inhibitors targeted against C-raf-1 kinase may be of considerable value as antineoplastic agents that display activity against a wide spectrum of tumor types at well-tolerated doses.

Animals↗
PubMed1998

Structure of a stereoregular phosphorothioate DNA/RNA duplex.

In this work, we present the first NMR solution structure of a DNA/RNA hybrid containing stereoregular Rp-phosphorothioate modifications of all DNA backbone linkages. The complex of the enzymatically synthesized phosphorothioate DNA octamer (all-Rp)-d(GCGTCAGG) and its complementary RNA r(CCUGACGC) was found to adopt an overall conformation within the A-form family. Most helical parameters and the sugar puckers of the DNA strand assume values intermediate between A- and B-form. The close structural similarity with the unmodified DNA/RNA hybrid of the same sequence may explain why both the natural and the sulfur-substituted complex can be recognized and digested by ribonuclease H.

Base Sequence↗
PubMed2005

Phosphorothioate-stimulated uptake of short interfering RNA by human cells.

The cellular delivery of short interfering RNA (siRNA) is a main hurdle in therapeutic drug development. Here, we describe that phosphorothioate (PTO)-derived oligonucleotides stimulate the physical cellular uptake of siRNA in trans in human cells. This is reflected by an apparent dose-dependent siRNA-mediated suppression of lamin A/C in primary human umbilical vein endothelial cells. The PTO-stimulated cellular uptake in trans is concentration dependent, length dependent, related to the phosphorothioate chemistry but not sequence specific. We provide experimental evidence to support a caveolin-mediated uptake mechanism. In sum, this work strongly suggests the exploration of PTOs as facilitators in the delivery of biologically active siRNA to mammalian cells.

Caveolins↗
PubMed2005

Mechanistic studies of La3+- and Zn2+-catalyzed methanolysis of aryl phosphate and phosphorothioate triesters. Development of artificial phosphotriesterase systems.

The methanolyses of a series of O,O-diethyl O-aryl phosphates (2,5) and O,O-diethyl S-aryl phosphorothioates (6) promoted by methoxide and two metal ion systems, (La3+)2(-OCH3)2 and 4:Zn2+:-OCH3 (4 = 1,5,9-triazacyclododecane) has been studied in methanol at 25 degrees C. Brønsted plots of the logk2 values vs. pKa for the phenol leaving groups give beta(lg) values of -0.70, -1.43 and -1.12 for the methanolysis of the phosphates and -0.63, -0.87 and -0.74 for the methanolysis of the phosphorothioates promoted by the methoxide, La3+ and Zn2+ systems respectively. The kinetic data for the metal-catalyzed reactions are analyzed in terms of a common mechanism where there is extensive cleavage of the P-XAr bond in the rate-limiting transition state. The relevance of these findings to the mechanism of action of the phosphotriesterase enzyme is discussed.

Catalysis↗
PubMed1985

The specificity of yeast low-Km cyclic AMP phosphodiesterase towards free bivalent metal ions and the diastereoisomers of cyclic adenosine phosphorothioate.

The relative activity of a zinc-containing cyclic AMP phosphodiesterase towards the (Sp)- compared with the (Rp)-diastereoisomer of cyclic adensine phosphorothioate varied with the identity of the free bivalent metal ion from more than 35 to 0.074 along the series Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Cd2+, showing that this ion, and not the tightly bound zinc, bonds to the phosphorothioate moiety of the substrate.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Inhibition of hepatic gluconeogenesis by the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The specific intracellular cyclic AMP-dependent protein kinase antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), inhibited both basal and cyclic AMP-agonist-induced rates of gluconeogenesis in hepatocytes isolated from fasted rats. Incubation of the cells in the presence of pyruvate and lactate and either the Sp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS) or glucagon produced a concentration-dependent increase in the rate of gluconeogenic glucose production which was shifted to higher concentrations of Sp-cAMPS or glucagon in the presence of Rp-cAMPS. Incubation of the cells with Rp-cAMPS in the absence of agonist produced no increase in the rate of glucose production and, in most cases, 100 microM-Rp-cAMPS resulted in 14-20% decrease in the substrate-stimulated rate of glucose production. Sp-cAMPS-induced gluconeogenesis was inhibited half-maximally at 1 microM-Rp-cAMPS and glucagon-induced gluconeogenesis was inhibited half-maximally at 12 microM-Rp-cAMPS. Approx. 10-15% of the inhibition of gluconeogenesis observed in the presence of Rp-cAMPS was due to conversion of glucose 6-phosphate to liver glycogen, consistent with Rp-cAMPS-induced reactivation of glycogen synthase. The remaining 85-90% inhibition of gluconeogenic glucose production resulted from the action of Rp-cAMPS on the cyclic AMP-sensitive enzymes controlling the rate of gluconeogenesis.

Animals↗
PubMed2003

Optimization of functional efficacy of phosphorothioate-modified oligonucleotides in a human CD8+ T-cell ex vivo expansion model.

Antisense oligodeoxyribonucleotides (ODNs) can specifically inhibit gene expression, but their application to fresh human CD8+ T cells is limited by poor spontaneous uptake (<2%). We have examined and optimized the uptake of phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) into these cells in an ex vivo expansion model. Optimal antisense treatments were found to be, for fresh CD8+ T cells, 1 micro m PS-ODNs complexed with lipofectin (LF), which resulted in 35% uptake and 10 micro m PS-ODNs in the absence of LF, for cultured cells, which resulted in 95% uptake. The delivered antisenses were functional, as determined by the inhibition of protein expression. In this respect, partially phosphorothioate-modified ODNs (PS-ODNs-P) were twice as effective as completely modified (PS-ODNs-C), and the antisense specific for the cap site showed the highest protein suppression of those tested (68%). Uptake mechanisms were also investigated. To our knowledge, this is the first optimization of the delivery of antisense oligonucleotides into human CD8+ T cells. This protocol could be used to study the function of a particular gene in cytotoxic T lymphocytes and also by those looking for a method to deliver short interfering RNA into cell lines to specifically suppress a gene of interest.

CD8-Positive T-Lymphocytes↗
PubMed1981

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.

Base Sequence↗
PubMed1988

Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus.

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus 1 (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The modifications of oligomers include replacement of backbone phosphodiester groups with phosphorothioates and various phosphoramidates. All oligomers were found to be active. Oligomers with complete replacement of phosphodiesters with phosphoramidate or phosphorothioate groups were more active at the micromolar range than were unmodified oligomers of the same sequence. In addition, modified and unmodified homooligonucleotides also showed inhibition of HIV-1 replication. It is suggested that different classes of oligonucleotides may inhibit HIV replication by different mechanisms.

Animals↗
PubMed1989

Phosphorothioate and cordycepin analogues of 2',5'-oligoadenylate: inhibition of human immunodeficiency virus type 1 reverse transcriptase and infection in vitro.

Natural antiviral activity can be mediated by the interferon-induced synthesis of 2',5'-oligoadenylates (2-5As) and subsequent RNase L activation by these molecules. Analogues of 2-5A that are biologically active and metabolically stable were synthesized and analyzed for antiviral activity against the human immunodeficiency virus type 1 (HIV-1). Replacement of the 3' hydroxyl group of the adenosine moieties of 2-5A with hydrogen atoms (i.e., cordycepin analogues of 2-5A) converted authentic 2-5A trimer into anti-HIV-1 agents in vitro. These cordycepin analogues of 2-5A also inhibited partially purified HIV-1 reverse transcriptase. Introduction of chirality into the 2',5'-phosphodiester internucleotide linkages or 5'-phosphate moieties of the 2-5A molecule (i.e., phosphorothioate analogues of 2-5A) converted authentic 2-5A into more potent inhibitors of HIV-1 reverse transcriptase. However, these phosphorothioate 2-5As demonstrated little or no anti-HIV-1 activity in vitro. Thus, some analogues of 2-5A may form a class of anti-HIV-1 drugs with possible pleiotropic activities that include activation of latent RNase L and inhibition of reverse transcription.

Adenine Nucleotides↗
PubMed1989

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

Antisense oligodeoxynucleotides, both the phosphorothioate analogues and unmodified oligomers of the same sequence, inhibit replication and expression of human immunodeficiency virus already growing in tissue cultures of MOLT-3 cells with much greater efficacy than do mismatched ("random") oligomers and homooligomers of the same length and with the same internucleotide modification. This preferential inhibitory effect is elicited in as short a time as 4-24 hr postinfection. Likewise, antisense oligomers exhibit greater inhibitory effects on human immunodeficiency virus in chronically infected cells than do mismatched oligomers and homooligomers. Phosphorothioate antisene oligomers are up to 100 times more potent than unmodified oligomers of the same sequence in these inhibitory assays. These results, in major respects, confirm and extend those recently published by Matsukura et al. [Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitsuza, H., Wong-Staal, F., Cohen, J. S. & Broder, S. (1989) Proc. Natl. Acad. Sci. USA 86, 4244-4248]. They also point out the importance of computer analysis of sequences though to be random but that in reality contain significant areas of likely hybridization, either to the viral genome or to the complementary DNA strand synthesized from it. They thus reinforce the concept that specific base pairing is a crucial feature of oligonucleotide inhibition of human immunodeficiency virus.

Antiviral Agents↗
PubMed1992

Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum.

Synthetic oligonucleotides and their chemical modifications have been shown to inhibit viral and cellular gene expression by sequence-specific antisense hybridization to target mRNAs. We now report that oligodeoxynucleotide phosphorothioates and their nuclease-resistant modifications are effective in micromolar and submicromolar concentrations against the growth of both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Parasitized human erythrocytes were found to be accessible to radioactively labeled oligodeoxynucleotides, whereas the uninfected erythrocytes did not permit any cellular entry of the same compounds. The dihydrofolate reductase-thymidylate synthase gene of P. falciparum was demonstrated to be a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered modified oligonucleotides. The antimalarial activities observed in vitro were identical for chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. The antimalarial activity of oligodeoxynucleotide phosphorothioates is related to sequence complementarity to certain regions of the plasmodial genome as well as to non-sequence-defined activities.

Animals↗
PubMed1994

Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides.

A 20-mer phosphorothioate oligodeoxynucleotide designed to hybridize to the AUG translation initiation codon of mRNA encoding murine protein kinase C-alpha (PKC-alpha) inhibits the expression of PKC-alpha both in vitro and in vivo. In mouse C127 mammary epithelial cells, the reduction in PKC-alpha mRNA expression was both dose and time dependent. The oligodeoxynucleotide exhibited an IC50 value of 100-200 nM and reduced PKC-alpha mRNA expression for up to 48 hr. This reduction was specific for PKC-alpha versus other PKC isozymes (delta, epsilon, and zeta) and completely dependent upon oligodeoxynucleotide sequence. When administered intraperitoneally in mice, the same oligodeoxynucleotide caused a dose-dependent, oligodeoxynucleotide sequence-dependent reduction of PKC-alpha mRNA in liver, with an IC50 value of 30-50 mg/kg of body weight. Inhibition of expression was 64 +/- 11% after a single 50-mg/kg dose. The expression of PKC-delta, epsilon, and zeta mRNA was unaffected by this treatment. The oligodeoxynucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery systems, although in vitro, the oligodeoxynucleotide required cationic liposomes for inhibition of PKC-alpha expression. This study demonstrates the utility of phosphorothioate oligodeoxynucleotides as specific inhibitors of gene expression in vivo after systemic administration.

3T3 Cells↗
PubMed1996

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

Base Sequence↗
PubMed1996

Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo.

To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C-raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C-raf gene expression in vitro, and antitumor activity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo. Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.

Animals↗
PubMed1996

Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates.

A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared. The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified. The Km and Vmax values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate-containing dodecamer. This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease. The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover. For further study, see the related article "Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone" (Wenz, A., Jeltsch, A., and Pingoud, A. (1996) J. Biol. Chem. 271, 5565-5573).

Base Sequence↗