Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “phosphorothioates”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed2006

Recent advances in the stereocontrolled synthesis of antisense phosphorothioates.

Antisense technology has advanced substantially in the past few decades and now is a well-established therapeutic approach in medicinal chemistry, and it may prove to be a valuable tool in the treatment of a wide range of diseases. Phosphorothioate oligonucleotides are among the most important and promising antisense agents. However, the key drawback lies in their polydiastereomerism, which manifests itself in the different chemical and biological properties of the diastereomeric species. Methodologies towards the stereocontrolled synthesis of antisense phosphorothioate oligonucleotides have been well investigated in recent years. In this review, the progress in this field is summarized.

Animals↗
PubMed1998

Structural requirements for cationic lipid mediated phosphorothioate oligonucleotides delivery to cells in culture.

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Base Sequence↗
PubMed2004

Effects of phosphorothioate anti-sense oligodeoxynucleotides on colorectal cancer cell growth and telomerase activity.

AIM: To investigate the inhibitory effect of phosphorothioate anti-sense oligodeoxynucleotides (PASODN) on colorectal cancer LS-174T cells in vitro and the mechanism of inhibition of telomerase activity in these cells. METHODS: PASODN were used to infect LS-174T cells and block human telomerase RNA (hTR) through anti-sense technology. The inhibitory effect of PASODN was evaluated by colony-forming inhibition assay and growth curve. Changes of telomerase activity in LS-174T cells were detected by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), and the level of apoptosis was analyzed by flow cytometry (FCM) assay. RESULTS: PASODN showed a dose and time-dependent inhibition of cell proliferation. The optimal dosage of PASODN was 10 mumol/L. The colony-forming efficiency was 10.3% in PASODN group after 10 d, whereas that in phosphorothioate mis-sense oligodeoxynucleotides (PMSODN) group with the same concentration and in PBS group (blank control) was 49.1% and 50.7%, respectively. PCR-ELISA results indicated that telomerase activity in the PASODN group was obviously inhibited in comparison with in the control groups (P<0.01, t = 3.317 and 3.241, t0.01(20) = 2.845). Meanwhile, before the number of cells was decreased, the morphological changes were observed in the cells of PASODN group. The cells in PASODN group showed the apoptotic peak at 72 h after infection, whereas the control group did not show. CONCLUSION: Specific sequence oligonucleotides can inhibit telomerase activity and lead to cell apoptosis, suggesting a novel treatment strategy for malignant tumors induced by telomerase.

Apoptosis↗
PubMed2000

Phosphorothioate backbone modification modulates macrophage activation by CpG DNA.

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

Adjuvants, Immunologic↗
PubMed1999

Phosphorothioate oligodeoxynucleotides promote the in vitro development of human allergen-specific CD4+ T cells into Th1 effectors.

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.

Adult↗
PubMed1999

Target selectivity of MAPK phosphorothioate antisense ODN on p42/p44, p38 MAPK, and JNK protein expression and its inhibitory effect on VSMC DNA synthesis.

AIM: To analyze the target selective and sequence-specific inhibitory effect of mitogen-activated protein kinase (MAPK) phosphorothioate antisense oligodeoxynucleotides (ODN) on p42/p44, p38 MAPK, c-jun NH2-terminal protein kinases (JNK) protein expression, and DNA synthesis in vascular smooth muscle cell (VSMC). METHODS: Using a phosphorothioate-protected 17-mer antisense MAPK ODN directed against the initiation of translation sites of the p42/p44 MAPK isoforms by liposomal transfection to deplete cultured rat, rabbit, and fetal calf VSMC MAP kinases. The 17-mer sense and random sequence MAPK ODN were used as controls. After liposomal transfection, cells were exposed to 20% serum for 24 h, and then harvested in lysis buffer. P42/p44, p38 MAPK, and p46/p58 JNK protein expression were measured by Western blot. DNA synthesis was measured by [3H]thymidine incorporation. RESULTS: Treatment with MAPK antisense ODN (0.1-0.8 mumol.L-1) for 48 h reduced phosphored p42/p44 MAPK protein expression but without effect on p38 MAPK and JNK expression, and inhibited cultured rat, rabbit, and fetal calf VSMC [3H]thymidine incorporation stimulated by 20% serum in a concentration-dependent manner. CONCLUSION: The MAPK antisense ODN target-selectively and sequence-specifically reduces the p42/p44 MAPK protein expression and concentration-dependently inhibits proliferation of rat, rabbit and fetal calf VSMC.

Animals↗
PubMed2000

[Study on the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide on coxsackie virus B replication in vitro].

OBJECTIVE: To investigate the antiviral effect of antisense phosphorothioate oligodeoxynucleotide (AODN) on CVB3 replication in vitro. METHODS: In this study, 21 mer antisense phosphorothioate oligodeoxynucleotide which complemented to nt 581-601 in 5' NCR of Coxsackie Virus B3 (CVB3) RNA was used to investigate antiviral activity in Vero cells as a specific inhibitor of CVB3 replication. Specific AODN of CVB3 RNA, sense oligodeoxynucleotide (SODN) and randomized nonsense-sequence RODN were synthesized for effect comparison among them. Vero cells infected by CVB3 were transfected with different concentrations of AODN mediated by lipofectamine reagent, so that the cells could take in more AODN. Cell control and viral control were set up. The inhibitory effect of AODN on CVB3 replication was evaluated with a number of variables, including inhibitory rates of cytopathic effect (CPE), cell survival rates by MTT assay, inhibitory rates of CVB3 antigen by ELISA, inhibitory rates of RNA by dot blotting and 50% tissue culture infective dose (TCID50). RESULTS: The specific AODN could significantly inhibit CPE of CVB3-infected Vero cells, decrease the production of antigen and RNA of CVB3 and viral titers, and increase cell survival rates, in a dose-dependent manner. The strongest inhibitory effect appeared at 48 hours after transfection, the most effective concentration of AODN was 10 micromol/L. On the other hand, 10 micromol/L SODN also showed weaker inhibitory effect on CPE of CVB3-infected cells, but no antiviral effect of 10 micromol/L RODN was shown. AODN showed no-inhibition on HSV-1 replication, but inhibited some enteroviruses such as CVB3, Polio-1 and Echo-6 to some extent. CONCLUSIONS: These results indicate that nt 581-601 in 5' NCR of CVB3 RNA may play an important role in regulating CVB3 replication and AODN may have inhibitory effect on CVB3 replication.

Animals↗
PubMed1992

Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.

Base Sequence↗
PubMed2005

Dimethylthiarum disulfide: new sulfur transfer reagent in phosphorothioates oligonucleotide synthesis.

Dimethylthiarum disulfide (DTD) has been developed as a new and efficient sulfur-transfer reagent for automated synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry. The traditional four-step automated oligonucleotide synthesis has been compressed to three-step protocol using DTD. This improvement allowed an overall 20% reduction in the solvent consumption and reduced the total synthesis time by 25%. The large-scale application of DTD has been successfully demonstrated by synthesis of therapeutically useful 20-mer phosphorothioate antisense oligonucleotides with excellent yield and purity.

Base Sequence↗
PubMed2004

[Effect and mechanism of VEGF antisense phosphorothioate deoxynucleotides on HL-60 leukemic cells].

To explore dose-effect or time-effect of vascular endothelial growth factor (VEGF) antisense phosphorothioate oligodeoxynucleotides (AS PS-ODN) on growth of HL-60 cells, and to study the effect mechanism so as to find new role of VEGF, A7, which was the most effective one of AS PS-ODN selected with computer-aided design and experimental assay, contains 20-DNA modified with phosphorothioate and was tranferred into cells mediated with lipofectin. After culture for 72 hours, inhibitive rate of cell growth was detected with MTT methods, viable cells were counted with trypan blue exclusion each 24 hour, cell configuration and apoptosis were observed with Geimsa staining and flow cytometry respectively, level of VEGF protein was detected with VEGF ELISA kit. The results showed that A7 is able to inhibit cell growth of HL-60 in dose-depending manner of AS PS-ODN, to down-regulate VEGF protein expression significantly, and not to induce apoptosis of HL-60 cells. It is concluded that there is possibility that the inhibition effect of VEGF AS PS-ODN on HL-60 cell growth is to restrain cell proliferation without inducing apoptosis of HL-60 cell, which would interpret that endogenous VEGF proteins have a capacity of promoting proliferation of HL-60 cell.

Cell Proliferation↗
PubMed1991

Synthetic phosphorothioate-containing analogues of inositol 1,4,5-trisphosphate mobilize intracellular Ca2+ stores and interact differentially with inositol 1,4,5-trisphosphate 5-phosphatase and 3-kinase.

Intracellular Ca2+ stores in permeabilized SH-SY5Y neuroblastoma cells were mobilized by D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] and two of its synthetic analogues, DL-myo-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3-5S) and DL-myo-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3). The concentrations of D-Ins(1,4,5)P3, DL-InsP3-5S, and DL-InsP3S3 required for half-maximal release were 0.11, 0.8, and 2.5 microM, respectively. All agents were full agonists, releasing 55-60% of sequestered 45Ca2+. D-Ins(1,4,5)P3-induced mobilization of Ca2+ was transient, and Ca2+ reuptake followed D-Ins(1,4,5)P3 metabolism closely. DL-InsP3S3-induced mobilization was persistent, consistent with the resistance of this analogue to metabolic enzymes. In contrast, DL-InsP3-5S-induced Ca2+ mobilization was followed by reuptake of Ca2+, albeit at a slower rate than that seen with D-Ins(1,4,5)P3. DL-InsP3-5S and DL-InsP3S3 were resistant to D-Ins(1,4,5)P3 5-phosphatase and potently inhibited the enzyme, with Ki values of 6.8 and 1.7 microM, respectively. DL-InsP3S3 was resistant to D-Ins(1,4,5)P3 3-kinase and was a very weak inhibitor of the enzyme (Ki = 230 microM). The ability of DL-InsP3-5S to inhibit D-Ins(1,4,5)P3 phosphorylation (apparent Ki = 5 microM) and its loss of Ca(2+)-releasing ability on incubation with D-Ins(1,4,5)P3 3-kinase suggest that this analogue may undergo phosphorylation to inositol 1,3,4-trisphosphate 5-phosphorothioate. These differential and complementary properties of DL-InsP3-5S and DL-InsP3S3 may be useful in dissecting the roles of D-Ins(1,4,5)P3 and D-myo-inositol 1,3,4,5-tetrakisphosphate in Ca2+ homeostasis.

Calcium↗
PubMed1991

Cell-free translation system using phosphorothioate-containing mRNA.

Phosphorothioate-containing RNAs were generated by transcription of template DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) (NTP alpha S) and T7 RNA polymerase. The substitution of mRNA by phosphorothioate increased the efficiency of protein synthesis by stabilizing the mRNAs in prokaryotic cell-free translation systems. The substituted mRNAs were also shown to be applicable to the continuous cell-free translation system developed by Spirin and coworkers.

Escherichia coli↗
PubMed1991

Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer.

In search of critical genes in the mechanism of estrogen action in human breast cancer, we previously showed that estrogen stimulates transcription of the c-myc gene in estrogen-dependent (MCF-7) cells. We have now examined the role of c-myc in estrogen-stimulated growth of MCF-7 cells through the use of a synthetic antisense c-myc phosphorothioate oligonucleotide to specifically inhibit expression of the c-myc protein. Estrogen induces a 5-fold increase in c-myc protein expression within 90 min in steroid-deprived cells, as detected by Western blot. Prior exposure of MCF-7 cells to 10 microM c-myc antisense oligonucleotide results in up to 95% inhibition of the c-myc protein expression induced by estrogen. Antisense-myc oligonucleotide inhibits estrogen-stimulated cell growth by up to 75% over 9 days and also exerts a cytostatic effect on the growth of estrogen-independent MDA-MB-231 cells which show relatively high, constitutive expression of c-myc. Sense-myc and antisense-pS2 oligonucleotides have no effect on c-myc protein level or growth in either cell line. These results demonstrate both the specific and durable effects of antisense phosphorothioate oligonucleotides. Furthermore, these results indicate a critical role for c-myc in the growth of breast cancer cells and support the hypothesis that loss of estrogen regulation of this gene may be an important factor in the progression of breast cancer.

Base Sequence↗
PubMed1979

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Biodistribution of the radioprotective drug 35S-labeled 3-amino-2-hydroxypropyl phosphorothioate (WR77913).

3-Amino-2-hydroxypropyl phosphorothioate (WR77913), a less toxic phosphorothioate radioprotector than WR2721, has been labeled with 35S. The biodistribution of a radioprotective dose of 800 mg/kg was determined in C3H mice bearing RIF-1 tumors as a function of time after intraperitoneal injection and was expressed as percentage injected dose/gram (% ID/g). Levels of 35S in the blood peaked 10 min after injection, and radioactivity in most tissues was highest at 15 min. Label in most tissues declined markedly between 15 and 60 min, but in gut, salivary glands, tumor, and brain, the levels of radioactivity remained quite stable over 1 hr. At 30 min after injection the highest levels of labeled drug were found in submandibular salivary glands, gut, and kidney, with the lowest level in brain. Tumors had approximately the same amount of label as blood, muscle, skin, and esophagus. Two principal differences between the distribution of label from WR77913 and WR2721 were defined. Although blood levels of 35S-WR2721 also peaked 10 min after injection, the 10-min blood levels achieved for WR77913 were more than fourfold greater than those attained by WR2721. Maximum levels of WR2721 occurred in most tissues 30 to 60 min after administration of the drug, compared to 15 min for WR77913. The basis for these differences remains to be determined, but these results suggest that the optimum interval between administration of WR77913 and irradiation may be shorter than for WR2721.

Amifostine↗
PubMed1986

Comparison of the protective effects of three phosphorothioate radioprotectors in the RIF-1 tumor.

Three aminoalkyl phosphorothioates, WR-2721, WR-3689, and WR-77913, were compared as radioprotectors of RIF-1 tumors irradiated in vivo and assayed for cell survival in vitro. The protector doses were 50% of the acute drug LD50. The radiation dose modifying factors for the three drugs were nearly equal, ranging from 1.5 to 1.7 at surviving fractions of 0.1 and 0.05. Using biodistribution data obtained with 35S labeled drugs, the uptake in tumors was calculated as micromoles drug per gram of tumor. On this basis, tumor levels of WR-77913 were 4.5-fold those of WR-2721, and WR-3689 uptake was 2.7-fold greater than uptake of WR-2721. Thus, on a molar basis, WR-2721 appears to be the most effective protector, but all three phosphorothioates protect this tumor moderately well. In diffusible substance autoradiographs of 3H WR-3689 labeled tumors, label was generally distributed over cells with no evidence of preferential localization over nuclei.

Amifostine↗
PubMed1982

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates.

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.

Adenosine Diphosphate↗
PubMed1995

Contribution of sequence and phosphorothioate content to inhibition of cell growth and adhesion caused by c-myc antisense oligomers.

c-myc is overexpressed in glioblastoma multiforme, the most common form of brain tumor. To find a suitable target for in vivo antisense therapy of gliomas, we investigated the biological effects on the human glioma cell line, U87MG, of antisense oligonucleotides targeted against the translation start site of c-myc mRNA. Parameters examined included c-myc protein level, cell proliferation, and cell adhesion to substratum. Oligonucleotides were administered by electroporation as capped phosphorothioates. Antisense oligomers caused a reduction in c-myc protein expression, loss of cell adhesion to plastic, and complete growth inhibition. Various control sequences, including sense, scrambled, and three-base mismatched oligomers, were also tested. Some of the controls retained a dG quartet found in the antisense sequence. Reduction in c-myc protein and cell growth and loss of cell adhesion were specific to the antisense sequence. Surprisingly, fully thioated antisense and scrambled sequences, either containing or lacking a dG quartet, were equally inhibitory to both cell growth and adhesion. Loss of cell adhesion was observed with only phosphorothioate-containing oligomers, not with either their phosphodiester or nuclease-resistant PA congeners, and was completely reversed when cells were plated onto fibronectin. These results demonstrate that a commonly used c-myc antisense oligomer also displays dramatic, sequence- but not antisense-specific effects on cell proliferation and cellular adhesion, depending on the backbone.

Animals↗