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PubMed1995

Toxic interactions between fungicides that inhibit ergosterol biosynthesis and phosphorothioate insecticides in the male rat and bobwhite quail (Colinus virginianus).

The potential for toxic interactions between ergosterol biosynthesis-inhibiting fungicides (EBIFs), used in U.S. agriculture or clinically, and phosphorothioate insecticides was assessed in adult male rats and adult male bobwhite quail (Colinus virginianus) by measuring inhibition of plasma butyryl cholinesterase (BChE) following fungicide and insecticide treatment. Male Sprague-Dawley rats (300 g) were administered corn oil or the following EBIFs: propiconazole (400 mg/kg/day), vinclozolin (400 mg/kg/day), clotrimazole (100 mg/kg/day), or ketoconazole (100 mg/kg/day) for 3 days by oral gavage. Forty-eight hours following the final dose, a single bolus of parathion (0.4 mg/kg in corn oil) or malathion (150 mg/kg in corn oil) or corn oil alone was given po. The rats were terminated 12 hr following parathion or 4 hr following malathion dosing. Significant (p < 0.05) inhibition of BChE was observed with parathion and malathion only following clotrimazole treatment. In contrast, when a similar experiment was performed in bobwhite quail dosed with 12 mg/kg malathion following EBIF treatment, significant BChE inhibition was observed following treatment with vinclozolin or ketoconazole, but not with propiconazole or clotrimazole. Induction of cytochrome P450 in rat and quail liver by EBIFs was accompanied by enhanced oxidative desulfuration of malathion, parathion, and diazinon to toxic oxon products. Increased detoxication via oxidative dearylation/esterolytic clevage also occurred. However, while enhanced acute in vivo insecticide toxicity was observed in both species with a number of EBIF-phosphorothioate combinations, EBIF-induced oxidative activation of phosphorothioates by liver microsomes in vitro was not a good predictor of this effect.

Administration, Oral↗
PubMed2004

Comparison of the cleavage profiles of oligonucleotide duplexes with or without phosphorothioate linkages by using a chemical nuclease probe.

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO(5) is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5'-GCAAAAGC/5'-GCTTTTGC duplexes by Mn-TMPyP/KHSO(5) was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all- Rp-phosphorothioate and all- Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)(3)-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.

Binding Sites↗
PubMed1992

Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates.

Oligodeoxynucleotide-containing phosphorothioate backbones have been used to regulate viral as well as cellular gene expression. The studies carried out in tissue culture have shown promising results on the use of oligonucleotide phosphorothioates as antiviral agents and, at present, study is underway to develop these oligonucleotide analogues as chemotherapeutic agents. To analyze and purify oligonucleotide analogues, high-performance liquid chromatography using weak anion exchange column has been described. The separation of oligonucleotide phosphorothioate is found to be length dependent.

Base Sequence↗
PubMed1989

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs.

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.

Animals↗
PubMed1985

Interaction of O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl phosphorodithioate, the impurities of malathion with supercoiled PM2 DNA.

The interaction of O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl phosphorodithioate, the impurities found in malathion, with DNA at pH 8.0, was investigated. Supercoiled PM2 DNA was incubated with these compounds at pH 8.0 at 37 degrees C and then the superhelicity of the modified DNA was determined by gel electrophoresis. Both compounds caused unwinding of supercoiled DNA in dose- and incubation time-dependent manner. O,S,S-trimethyl phosphorodithioate was a more potent agent than O,O,S-trimethyl phosphorothioate. At 37 degrees C following 2.0 hours incubation, 100 mM O,S,S-trimethyl phosphorodithioate produced fully unwound DNA, whereas at 200 mM O,O,S-trimethyl phosphorothioate produced 80% unwound DNA following 12 hours' incubation. At the same condition, 5 mM methyl methanesulfonate, a potent alkylating mutagen, produced fully unwound DNA following 1 hour incubation at 5 mM. These results indicated that there were chemical interactions between these agents and DNA. The possibility of the interaction of OOS-TMP being as a covalent intercalation as well as strand nicking was discussed.

Animals↗
PubMed1991

Clearance of S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913) in rats.

Complications of radiotherapy in the treatment of retinal and choroidal neoplastic diseases include cataract formation, radiation retinopathy, neovascular glaucoma, cystoid macular edema, and subretinal neovascularization. These side effects may be minimized by the use of compounds known to have a protective effect on normal ocular tissues without impeding the benefits of therapy. Phosphorothioates, first developed under the Antiradiation Drug Development Program of the U.S. Army Medical Research and Development Command, have been reported to protect normal tissues during radiation therapy in a variety of animal models. One of the phosphorothioates, WR-77913 (S-[3-amino-2-hydroxylpropyl]phosphorothioate) was found to inhibit cataract formation in rats after radiation exposure. To test the efficacy of WR-77913 in the retina, we established a high-pressure liquid chromatography method to measure the levels of dephosphorylated WR-77913 and studied the drug's clearance from the lens, retina, blood, kidney, and liver in rats.

Amifostine↗
PubMed1995

Inhibition of HIV-1 in vitro by C-5 propyne phosphorothioate antisense to rev.

A 15-mer C-5 propyne modified phosphorothioate oligodeoxynucleotide antisense to rev was approximately 5-fold more effective in providing viral inhibition compared to a 28-mer unmodified phosphorothioate oligodeoxynucleotide targeted to the same sequence and previously shown to inhibit HIV-1 in a sequence-dependent manner. The antiviral effect was obtained by lipofection or simple addition of 0.2-1 microM modified oligodeoxynucleotide to the culture medium of H9 cells chronically infected with the HIV-1LAI isolate of human immunodeficiency virus type 1. We conclude that C-5 propyne oligodeoxynucleotides in accordance with previous findings by others are superior to unmodified phosphorothioates in providing inhibition of HIV-1 in a sequence-dependent manner and that this inhibition can be conferred by oligodeoxynucleotides in free solution.

Antiviral Agents↗
PubMed1993

Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy.

Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI). The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme. The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments. In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide. The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al. (1989) Biochemistry 28, 1340). The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C). Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme.

Base Sequence↗
PubMed1988

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.

Antiviral Agents↗
PubMed1995

Antiviral effect of phosphorothioate oligodeoxyribonucleotides complementary to human immunodeficiency virus.

Modifications of oligodeoxyribonucleotides include the replacement of the backbone phosphodiester groups with phosphorothioate (S-ODNs) groups and the substitution of phosphorothioate (SO-ODNs) groups at both the 3'- and 5'-ends. In assays for HIV, oligomers (S-ODNs) were more active at the micromolar range than were SO-ODNs of the same sequence. Furthermore, the abilities of antisense-, sense-, random-, and mismatched-oligomers, or homo-oligomers containing internucleotidic phosphorothioate linkages to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-, sense-, mismatched-, and homo-oligomers of the same length or with the same internucleotide modification. Five different target sites (gag, pol, rev, tat, and tar) within the HIV genes were also studied with regard to the inhibition of HIV replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of initiation sequences and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting viral replication was also investigated. Of particular interest was the S-ODNs-rev 15 mer, which possessed higher anti-HIV activity than the sense-, random-, mismatched-, and homo-20 mers.

Antiviral Agents↗
PubMed2005

High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer.

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.

DNA Primers↗
PubMed2004

CG sequence- and phosphorothioate backbone modification-dependent activation of the NF-kappaB-responsive gene expression by CpG-oligodeoxynucleotides in human RPMI 8226 B cells.

Oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention because of their stimulatory effects on innate immune responses. CpG-ODN 1826 containing two GACGTT motifs is well known to activate the mouse immune cells while CpG-ODN 2006 containing three GTCGTT motifs is optimal for human cells. We have shown that stimulation of the human B cell line RPMI 8226 with CpG-ODN 1826 or 2006 results in the activation of IL-8 promoter and nuclear localization of NF-kappaB in the CG sequence- and phosphorothioate backbone modification-dependent manner. It was also demonstrated that myeloid differentiation protein and tumor necrosis factor receptor-associated factor 6 are involved in the signal transduction pathway triggered by the CpG-ODNs. Furthermore, phosphorothioate-modified CpG-ODN 1826 led to induce the NF-kappaB-responsive inflammatory cytokine gene expression in the cells. Experimental results indicated that the phosphorothioate derivative of CpG-ODN 1826 not only activates the mouse immune cells, but also stimulates NF-kappaB responsive gene expression in the human B cell line.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Binding of phosphorothioate oligonucleotides to zwitterionic liposomes.

Although double-stranded DNA (dsDNA) has been shown to bind to zwitterionic lipids, it has been reported that this association is stronger for disordered (L(alpha)) phase lipids than for well-ordered (L(beta)) lipids. In this work, the interaction of single-strand phosphorothioate oligonucleotides (ONs) with unilamellar liposomes of saturated and unsaturated zwitterionic phosphocholines (PCs) and phosphoroethylamine (PE) was investigated. It is shown that the association of phosphorothioate ONs to diacyl glycerophosphocholines is strong, but only for L(beta) phase or otherwise ordered bilayers. There is no measurable affinity for PE lipids. The apparent affinity of three different phosphorothioate ONs for L(beta) phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been measured and the dissociation constants were on the order of 10(-7) M. Purine-rich ON sequences had stronger binding to DPPC liposomes than did pyrimidine-rich sequences, but there were other sequence-dependent factors. This exceptionally high affinity could be an important consideration in ON uptake, delivery, and biodistribution.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Selective binding of trisamine-modified phosphorothioate antisense DNA to target mRNA improves antisense activity and reduces toxicity.

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature (T(m)) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.

Anti-HIV Agents↗
PubMed1988

RNA mediated formation of a phosphorothioate diester bond.

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

Base Sequence↗
PubMed2001

Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein.

The efficacy of antisense oligonucleotides depends on the ability to reach in vivo their target cells. We aim to develop strategies to enhance uptake of phosphorothioate oligodeoxynucleotides by Kupffer cells. To this end, we conjugated cholesterol to ISIS-3082, a phosphorothioate oligodeoxynucleotide specific for intercellular adhesion molecule-1. The cholesterol-conjugated oligonucleotide, denoted ISIS-9388, associated readily with lactosylated low-density lipoprotein (LacLDL), a lipidic carrier that is taken up by galactose receptors on Kupffer cells. Association of up to 10 molecules of ISIS-9388 per LacLDL particle did not induce aggregation. LacLDL-associated [3H]ISIS-9388 was rapidly taken up by the liver after injection into rats (52.9+/-1.8% of the dose within 2 min versus 18.6+/-2.8% for ISIS-3082). N-acetylgalactosamine inhibited hepatic uptake, indicating involvement of galactose-specific receptors. Liver cells were isolated at 60 min after injection of LacLDL-associated [3H]ISIS-9388. Kupffer cells displayed the highest uptake: 88.1+/-24.7 ng of oligonucleotide/mg of cell protein, which is 6-14 times higher than after injection of free ISIS-9388 or ISIS-3082 (15.0+/-3.8 ng and 6.3+/-1.4 ng, respectively). It can be calculated that Kupffer cells contribute 43.9+/-5.4% to the liver uptake (free ISIS-9388 and ISIS-3083 14.5+/-3.1% and 8.3+/-3.2%, respectively). In conclusion, conjugation of a phosphorothioate oligodeoxynucleotide with cholesterol and its subsequent association with LacLDL results in a substantially increased Kupffer cell uptake of the oligonucleotide. As Kupffer cells play a key role in inflammation, our approach may be utilized to improve antisense-based therapeutic intervention during inflammation.

Animals↗
PubMed1999

Omega-6 polyunsaturated fatty acid-stimulated cellular internalization of phosphorothioate oligodeoxynucleotides: evidence for protein kinase C-zeta dependency.

The rate of cellular internalization of phosphorothioate oligodeoxynucleotides is determined predominantly by adsorptive plus fluid-phase endocytosis. Internalization of a 5'-fluoresceinated phosphorothioate 15mer homopolymer of thymidine (FSdT15) in K562 cells in medium containing lipid-depleted albumin was reduced consistently versus nondepleted albumin. Treatment of K562 and several other cell lines with omega-6 polyunsaturated fatty acids (omega-6 PUFAs; e.g. arachidonic and linoleic acids) but not saturated fatty acids dramatically increased FSdT15 internalization in a concentration-dependent manner and over a wide albumin concentration range. The rate of efflux of FSdT15 from K562 cells was not affected by the omega-6 PUFA, implying that an increase of cellular fluorescence was due to an increase in the in-rate. These data were consistent with the observation that the binding of FSdT15 to the cell surface was also increased in the presence of omega-6 PUFAs. Omega-6 PUFAs are stimulators of protein kinase C (PKC) activity. Inhibition of PKC activity in K562 cells by Go6976, an inhibitor of the classical PKC isoforms, did not block the linoleic acid-induced stimulation of FSdT15 internalization. On the other hand, treatment of cells with Ro318220, which has considerably less isoform specificity, almost totally blocked the effect of linoleic acid on FSdT15 internalization, implying the involvement of a nonclassical PKC isoform in the process. Finally, since the only PKC isoform expressed in K562 cells that also is activated by omega-PUFAs is PKC-zeta, we obtained NIH 3T3 cells expressing a doxycycline-repressible dominant negative PKC-zeta mutant. Expression of the mutant blocked the stimulation of FSdT15 internalization by linoleic acid. Stimulated internalization also was blocked by wortmannin and LY 294002, which are relatively specific inhibitors of phosphatidylinositol 3-kinase (PI 3-K). Taken together, our data suggest that omega-6 PUFA stimulation of fluoresceinated phosphorothioate oligomers may be PKC-zeta dependent, and perhaps PI-3K dependent as well.

Ceramides↗
PubMed1996

Amplification of antibody production by phosphorothioate oligodeoxynucleotides.

A phosphorothioate oligodeoxynucleotide that is complementary (antisense) to the initiation region of the rev gene of HIV-1 causes hypergammaglobulinemia and splenomegaly in mice, and it induces B cell proliferation and differentiation in mouse spleen mononuclear cells (SMNCs) and human peripheral blood mononuclear cells in vitro. The current studies were performed to investigate the specificity of these immunomodulatory effects. Both the sense and antisense rev oligomers stimulated tritiated thymidine incorporation and secretion of immunoglobulin M (IgM) and immunoglobulin G (IgG) by mouse SMNCs in a concentration-dependent fashion, but the antisense oligomer produced greater immune effects. Studies comparing phosphorothioate oligomers (anti-rev, c-myc, and c-myb) either methylated or unmethylated at CpG dinucleotides showed that methylation effectively abrogated the proliferative effect and tended to reduce the immunoglobulin secretory activity, but the latter was not statistically significant except in the case of IgG in anti-rev oligomer-treated cultures. Mice were injected with the sense or antisense rev oligomers singly or in combination. The animals then were immunized with tetanus toxoid and received a booster 21 days later. Oligodeoxynucleotide-treated mice had significantly higher levels of IgM antibodies on days 28 and 35 and of IgG antibodies on days 14 and 35 as compared with mice that were immunized but received vehicle alone. There was no evidence for additive, synergistic, or antagonistic interactions of the sense and antisense rev oligomers. These results indicate that the unmethylated anti-rev oligomer is the most potent of the phosphorothioate oligomers tested at activating lymphocyte proliferation and differentiation and that a single intravenous injection of this oligodeoxynucleotide augments antibody production to a specific antigen as long as 35 days later.

Animals↗