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PubMed1985

Trialkyl phosphorothioates and glutathione S-transferases.

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.

Animals↗
PubMed2001

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides.

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.

CD4 Antigens↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 replication by P-stereodefined oligo(nucleoside phosphorothioate)s in a long-term infection model.

Oligo(nucleoside phosphorothioate)s (S-ODNs), if prepared by conventional methods, consist of a mixture of diastereomers by virtue of the asymmetry of the phosphorus atom involved in the internucleotide linkages. This may affect the stability of the complexes formed between S-ODNs and complementary oligoribonucleotides, which is commonly accepted as the most important factor in determining the efficacy of an antisense approach. Using HIV-1-infected MOLT-4 cells via a long-term culture approach, we studied the influence of the P-chirality sense of stereodefined 28mer oligo(nucleoside phosphorothioate)s, [All-Rp]-S-ODN-gag-28-AUG and [All-Sp]-S-ODN-gag-28-AUG, complementary to the sequence starting at the AUG initiation codon of the gag mRNA of HIV-1, upon the anti-HIV-1 activity. The [All-Sp]-S-ODN-gag-28-AUG at a low concentration of 0.5 microM can completely suppress HIV-1(gag) p24 antigen expression in HIV-1-infected MOLT-4 clone 8 cells for 32 days. Cells treated with [All-Rp]-S-ODN-gag-28-AUG (0.5 microM) showed a high level of the antigen expression at day 16. Furthermore, satisfactory suppression could not be achieved from a random [Mix]-S-ODN-gag-28-AUG, consisting of a diastereomeric mixture of the oligonucleotides. Our results suggest that chemotherapy based upon the use of stereodefined antisense [All-Sp] S-ODN may be a more effective method for reducing the viral burden in HIV-1-infected individuals.

Anti-HIV Agents↗
PubMed1985

Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other backbone-modified congeners of DNA.

Diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides, which were synthesized by an automated, solid-phase, phosphoramidite-coupling method, were conveniently separated by reversed-phase high-performance liquid chromatography on a muBondapak C18 column with a gradient of acetonitrile in triethylammonium acetate buffer. These synthetic and chromatographic methods were also used to obtain diastereomerically pure bis-phosphorothioate, alkanephosphonate, and O-alkyl phosphotriester analogues of DNA for exploratory studies of stereochemistry and phosphorolytic enzymes.

Chemical Phenomena↗
PubMed2000

Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity.

The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3' end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3' end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.

Bacteriophage mu↗
PubMed1998

The binding of anti-DNA antibodies to phosphorothioate oligonucleotides in a solid phase immunoassay.

Phosphorothioate oligonucleotides (S-oligos) are nucleic acid derivatives that are commonly used as antisense agents. These compounds, similar to bacterial DNA and CpG oligonucleotides, display a variety of immunological activities in vitro and in vivo. To assess further these activities, the antigenicity of a series of S-oligos was assessed in the solid phase using anti-DNA antibodies from sera of patients with systemic lupus erythematosus. By ELISA, S-oligos bound well to anti-DNA antibodies under the same conditions as calf thymus DNA antigen. The specificity for anti-DNA was established by competition assays showing cross-inhibition of binding by DNA and S-oligos. Reactivity with anti-DNA was observed with S-oligos varying in sequence, suggesting interaction with a conserved determinant not strictly dependent on the bases. Furthermore. in comparison with a phosphodiester oligomer of the same sequence, a phosphorothioate showed dramatically increased activity. These findings indicate that structural features associated with the S-oligo backbone promote specific binding to anti-DNA antibodies and influence the size requirement for antigenicity in the solid phase. These observations thus extend the immunological properties of S-oligos and suggest uses of these compounds in the diagnosis and treatment of autoimmune disease.

Animals↗
PubMed1979

Specific heavy metal labeling of the 3-'terminus of phosphorothioate modified yeast tRNAPhe.

Yeast tRNAPhe containing a phosphorothioate modified -CS-CS-A terminus binds two moles of chloroterpyridineplatinum(II). This result was determined by titrating the tRNA with [3H](terpy)PtCl] Cl, removing excess platinum by cation exchange chromatography, and determining the amount of bound platinum by radiocounting techniques. It has thus been established that adjacent phosphorothioate modified nucleotides can be labeled with an electron dense stain, a necessary requirement for electronmicroscopic sequencing of polynucleotides to become practical.

Kinetics↗
PubMed1998

Use of T7 gene 6 exonuclease and phosphorothioated primers for the manipulation of HIV-1 infectious clones.

A method is described for the efficient substitution, deletion or insertion of any desired DNA sequence into any viral infectious clones without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction combined with the resistance of 2'-deoxynucleotides 5'-O-(1-thiotriphosphate) dNTPs [S] bonds (phosphorothiate bonds) to the 5'-3' double strand specific T7 gene 6 exonuclease (T7 Exo) digestion. Primers used to amplify the DNA target regions being manipulated present three phosphorothioate bonds from the fifteenth base at the 5' end. The enzyme activity was shown to be completely inhibited by the presence of more than one phosphorothioate residue at the 5' end of the DNA molecules. When the amplification products are submitted to the exonuclease digestion the hydrolytic T7 Exo activity generates a short single strand DNA tail which contains the nucleotide integrity of the 3' strand. Since the ends of two independently amplified products overlap they can regenerate a stable recombinant structure when further combined in the same reaction tube in the presence of T4 DNA ligase. This new method can be used for manipulating an HIV-1 full-length clone belonging to subtype D replacing the env (gp120) gene for an F subtype sequence.

Bacteriophage T7↗
PubMed1997

End-capped antisense oligodeoxynucleotides effectively inhibit gene expression in vivo and offer a low-toxicity alternative to fully modified phosphorothioate oligodeoxynucleotides.

Sulfur modification of oligodeoxynucleotides produces nuclease resistance but also leads to toxic effects when these compounds are administered in vivo. To assess their potential as viable alternatives to full phosphorothioate derivatives, we have used rotational behavior and immunohistochemistry to investigate the efficacy and longevity of phosphorothioate, end-capped antisense oligodeoxynucleotides in suppression of c-fos and ngfi-a in the striatum of adult rats. Our results suggest that, despite having a limited duration of action, these end-capped, chimeric oligodeoxynucleotides are capable of specifically inhibiting gene expression in vivo and may, therefore, possess broader application potential in chronic suppression models as the reduction of sulfur content is likely to greatly minimize their toxic effects.

Animals↗
PubMedDate not supplied

Kinetic analyses of the microsomal biotransformation of the phosphorothioate insecticides chlorpyrifos and parathion.

Chlorpyrifos [0,0-diethyl-0-(3,5,6-trichloro-2-pyridyl) phosphorothioate] was metabolized to chlorpyrifos oxon [0,0-diethyl-0-(3,5,6-trichloro-2-pyridyl) phosphate] and to 3,5,6-trichloro-2-pyridinol by mouse hepatic microsomes. Formation of both chlorpyrifos oxon and 3,5,6-trichloro-2-pyridinol required NADPH, and was inhibited by carbon monoxide. Kinetic analyses using direct linear plots determined the appKm's for formation of chlorpyrifos oxon and 3,5,6-trichloro-2-pyridinol to be 20.9 +/- 3.3 microM and 16.1 +/- 3.4 microM respectively, while the appVmax's for the same reactions were 3.9 +/- 0.2 nmols/100 mg liver/min and 8.1 +/- 0.3 nmols/100 mg liver/min respectively. Incubation of parathion [0,0-diethyl-0-(4-nitrophenyl) phosphorothioate] with mouse hepatic microsomes produced paraoxon [0,0-diethyl-0-(4-nitrophenyl) phosphate] and p-nitrophenol. The appKm's for the formation of paraoxon and p-nitrophenol were 29.6 +/- 4.2 microM and 26.5 +/- 3.8 microM respectively, with appVmax's of 5.8 +/- 0.6 nmols/100 mg liver/min and 6.7 +/- 0.5 nmols/100 mg liver/min, respectively. Incubation of both parathion and chlorpyrifos at various concentrations with mouse hepatic microsomes resulted in inhibition of production of paraoxon, p-nitrophenol, chlorpyrifos oxon, and 3,5,6-trichloro-2-pyridinol, which was characteristic of mixed type inhibition. This complex kinetic behavior could arise as a result of competitive interactions of parathion and chlorpyrifos with multiple forms of microsomal cytochrome P-450.

Animals↗
PubMed1998

Study of phosphorothioate-modified oligonucleotide resistance to 3'-exonuclease using capillary electrophoresis.

The effect of phosphorothioate (PS) internucleotide linkages on the stability of phosphodiester oligodeoxyribonucleotides (ODNs) was investigated using 25-mer ODNs containing single or multiple PS backbone modifications. The in vitro stability of the oligomers was measured both in 3'-exonuclease solution and in plasma. For the separation of ODNs, capillary electrophoresis with a replaceable polymer separation matrix was used. As expected, DNA fragments with PS linkages at the 3'-end were found to be more resistant to 3'-exonuclease hydrolysis. Also increasing exonuclease resistance was the non-specific adsorption of phosphorothioate ODNs to enzyme.

Electrophoresis, Capillary↗
PubMed1999

Phosphorothioate oligodeoxynucleotides can selectively alter neuronal activity in the cochlea.

A growing body of evidence indicates that extracellular adenosine triphosphate (ATP) may have a major role in cochlear function. Antagonists of ionotropic ATP receptors (P2X2) have significant effects on cochlear potentials and distortion product otoacoustic emissions (DPOAEs). We tested whether antisense oligodeoxynucleotides (ODNs) would mimic the functional deficiencies induced by the ATP antagonists through binding to P2X2 ATP receptor mRNA and thereby reduce the number of ATP receptors expressed in the membrane of the cells. Both a phosphorothioate ODN (S-ODN) antisense and a phosphodiester ODN (P-ODN) antisense to the P2X2 sequence and random sense ODNs containing 21 nucleotides were administered chronically (7 days) to the guinea pig cochlea via the perilymph compartment. Sound evoked cochlear potentials (cochlear microphonic; summating potential; compound action potential of the auditory nerve, CAP; latency of the first negative peak in the CAP, N1 latency) and DPOAEs were monitored to assess the effects of the ODNs. Results indicate that the phosphorothioate derivatives of both the antisense and random sense ODNs suppressed the CAP and prolonged the N1 latency with no significant effect on the other parameters. The P-ODNs had no effect. Since both the antisense and random sense S-ODNs had the same effect, we conclude that the S-ODNs affected neuronal function in a manner that did not involve binding to the ATP receptor mRNA.

Adenosine Triphosphate↗
PubMed2000

A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides.

Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA. To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared. The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer. Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene. Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method. Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol). Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL. In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1). The polymeric systems were ineffective. The effect of the complexation medium was further studied on CV-1 cells. Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM). Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size. Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes. In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides.

Cell Line↗
PubMed2001

Stereocontrolled synthesis of LNA dinucleoside phosphorothioate by the oxathiaphospholane approach.

The application of the oxathiaphospholane approach for the stereocontrolled synthesis of LNA dinucleoside phosphorothioate is described. The reaction of ring opening condensation proceeds in CH3CN solution in high yield and with over 96% stereoselectivity. One of diastereomers of LNA dinucleoside phosphorothioate (presumably R(P)) was found to be readily digested by svPDE.

Chromatography, High Pressure Liquid↗
PubMed2003

A novel solid support for synthesis of oligonucleotide 3'-phosphorothioate monoesters.

A new reagent immobilized on solid support allowing for solid-phase synthesis of oligonucleotides with a 3'-terminal phosphorothioate monoester is described. The support is compatible with phosphoramidite chemistry for automated oligonucleotide synthesis. Final deprotection with ammonia under standard conditions leads to oligonucleotide 3'-terminal phosphorothioate.

Ammonia↗
PubMed2001

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups.

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.

Amines↗
PubMed2000

Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties.

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.

Animals↗