Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

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.

Inhibition of telomerase by 2′-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells

2′-O-(2-methoxyethyl) (2′-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2′-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC50 values of 5–10 nM at 37°C. IC50 values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.

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.

Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA.

DNA and RNA are the polynucleotides known to carry genetic information in life. Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important. This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo. Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1. Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones. Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA. All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA. Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo.

Cutting edge: Species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified ohgonucleotides

Different DNA motifs are required for optimal stimulation Of mouse and human immune cells by CpG oligode-oxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CPG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 AWK than optimal motifs. When the CpG dinucleotide was inverted to GC In each ODN some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may he responsible for mediating many published CpG-independent responses to PS-ODN.

A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal growth factor receptor in A431 cells

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense mechanism affecting receptor tyrosine kinase activity.

Properties of acetylcholinesterase from Pisum sativum

Acetylcholinesterase (AChE) from Pisum sativum purified 28 fold showed two closely moving protein bands on polyacrylamide gel electrophoresis, both of which have AChE activity. AChE activity occurs in roots, stem and leaves, that in roots varying with age. Activity is optimal at pH 9 and at 30”. The energy of activation is 9.82 x lo3 J per mol and MW is greater than 200000. Although the enzyme can hydrolyze both choline and non-choline esters, it has greater affinity for acetylthiocholine (ATCh) and acetylcholine (ACh). ATCh inhibits the enzyme at higher concentrations and the K, is 0.2 mM with this as substrate. The enzyme is not as sensitive to Eserine as it is to Neostigmine. It is also inhibited by organophosphorus pesticides such as Fensulfothion, Parathion and Dimethoate. Treatment of the seeds with Fensulfothion [O, O-diethyl (p-methylsulfinylphenyl) phosphorothioate] affects growth and secondary root development. This might be explained by its inhibition of AChE and the consequent increase of endogenous levels of ACh.

Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study.

Duchenne muscular dystrophy is caused by dystrophin deficiency and muscle deterioration and preferentially affects boys. Antisense-oligonucleotide-induced exon skipping allows synthesis of partially functional dystrophin. We investigated the efficacy and safety of drisapersen, a 2'-O-methyl-phosphorothioate antisense oligonucleotide, given for 48 weeks.

Phase II study of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C alpha, in patients with previously treated low-grade non-Hodgkin's lymphoma

Background: The purpose of this study was to assess the efficacy and safety of ISIS 3521, an antisense phosphorothioate oligonucleotide to protein kinase C in patients with relapsed low-grade non-Hodgkin's lymphoma (NHL). Patients and methods: Twenty-six patients received ISIS 3521 (2 mg/kg/day) as a continuous infusion over 21 days of each 28-day cycle. Results: The median age of the patients was 53 years (range 37–77). Histological subtypes were low-grade follicular lymphoma (n=22) and B-cell small lymphocytic lymphoma (n=4). Twenty-one (81%) had stage III/IV disease. The median number of previous lines of chemotherapy was two (range one to six). A total of 87 cycles of ISIS 3521 were administered. Twenty-three patients were assessable for response. Three patients achieved a partial response. No complete responses were observed. Ten patients had stable disease. Grade 3–4 toxicity was as follows: neutropenia (3.8%) and thrombocytopenia (26.9%). Conclusions: ISIS 3521 has demonstrated anti-tumour activity in patients with relapsed low-grade NHL. There may be a potential role for this agent in combination with conventional chemotherapy for advanced low-grade lymphoma, and further trials are warranted.

SYNTHESIS AND PROPERTIES OF DITHYMIDINE PHOSPHATE ANALOGS CONTAINING 3'-THIOTHYMIDINE

Dithymidine-3'-S-phosphorothioate (d(TspT)) has been prepared from a 5'-O-monomethoxytritylthymidine-3'-S- phosphorothioamidite (7) by activation with 5-(p- nitrophenyl)tetrazole in the presence of 3'-O- acetylthymidine. The resulting dinucleoside phosphorothioite is readily oxidised to the corresponding 3'-S-phosphorothioate using either tetrabutylammonium (TBA) perlodate or TBA oxone and has been deprotected under standard conditions to yield d(TspT). This dithymidine phosphate analogue is comparatively resistant to hydrolysis by nuclease P1, but the P-S bond is readily cleaved by aqueous solutions of either iodine or silver nitrate. Dithymidine-3'-S-phosphorodithioate (d[Tsp(s)T] was prepared in an analogous fashion using sulphur to oxidise the intermediate dinucleoside phosphoro thiolte. Absolute stereochemistry has been assigned to the diastereoisomers of d by comparing their physical and chemical properties to those of the dinucleoside phosphorothioates.

Implication des endothélines et de leurs récepteurs vasculaires dans la circulation pulmonaire en condition contrôle et pathophysiologique

Le système endothéline (ET) est activé en condition d’hypertension pulmonaire (HTP). L’efficacité des antagonistes des récepteurs à l’ET a clairement été démontrée et a menée à l’approbation clinique de tels antagonistes dans le traitement de l’hypertension artérielle pulmonaire (HTAP). Toutefois, il existe présentement un important débat opposant l’utilisation d’un antagoniste sélectif des récepteur ETA à l’utilisation d’un antagoniste double ETA/ETB dans le traitement de cette pathologie. Bien que nous sachions que le système ET est activé et contribue à l’HTAP, les modifications locales de ce système induites par la pathologie, particulièrement au niveau des artères de résistance pulmonaires, demeurent inconnues. De plus, l’impact de ces modifications sur la réponse pharmacologique aux divers antagonistes des récepteurs à l’ET (sélectifs versus double) est d’une importance capitale. Ainsi, le but de la première étude de cette thèse était d’évaluer les modifications potentielles de la pharmacologie du système ET au niveau des artères de résistance pulmonaires induites par l’HTAP. Dans cette étude, nous avons démontré qu’en condition contrôle l’antagoniste sélectif ETA et l’antagoniste double n’ont eu aucun effet sur la réponse vasoconstrictrice à l’ET-1. Toutefois, en condition d’HTAP, les antagonistes sélectif et double ont tous deux été en mesure de réduire la vasoconstriction pulmonaire induite par l’ET-1. Une diminution importante de l’expression génique du récepteur ETB pourrait être à l’origine de cette modification du profil pharmacologique des antagonistes. Une meilleure compréhension des rôles joués par les récepteurs ETA et ETB au niveau des artères de résistance pulmonaires pourrait permettre l’optimisation des traitements de l’HTAP. Ainsi, le but de la deuxième étude était d’évaluer les effets d’un traitement antisens ex vivo dirigé contre l’ARNm des récepteurs ETA et ETB dans la vasoconstriction des artères de résistance pulmonaires induite par l’ET-1. Dans cette étude, nous avons démontré dans un premier temps que les récepteurs ETA et ETB pouvaient former des dimères au niveau des artères de résistance pulmonaires. De plus, nous avons observé qu’une réduction de l’expression protéique du R-ETA entraînait une potentialisation de la vasoconstriction ETB dépendante suggérant ainsi qu’en condition contrôle, le récepteur ETA aurait un effet inhibiteur sur la vasoconstriction pulmonaire induite par la stimulation du récepteur ETB. Les effets délétères de l’ET-1 sur la circulation pulmonaire sont bien connus, toutefois seules quelques études ont porté leur attention sur l’implication de l’ET-3 dans l’HTAP. Ainsi, le but de la troisième étude était d’évaluer l’implication potentielle de l’ET-3 dans l’HTAP. Dans cette étude, nous avons démontré qu’il était nécessaire en condition contrôle de bloquer simultanément les récepteurs ETA et ETB afin de réduire la réponse vasoconstrictrice pulmonaire à l’ET-3. En condition d’HTAP, nous avons observé une augmentation non-significative des concentrations plasmatiques d’ET-3 ainsi qu’une modification du profil pharmacologique des antagonistes des récepteurs à l’ET. En effet, l’utilisation de l’antagoniste sélectif ETA ou de l’antagoniste double était dans les deux cas en mesure de réduire la vasoconstriction pulmonaire à l’ET-3. Les résultats de ces trois études suggèrent qu’il est préférable d’utiliser un antagoniste double dans le traitement de l’HTAP. En effet, (1) en condition d’HTAP, l’utilisation d’un antagoniste double est aussi efficace que l’utilisation d’un antagoniste sélectif ETA; (2) les récepteurs ETA et ETB peuvent former des dimères au niveau des artères de résistance pulmonaires et (3) le récepteur ETB joue un rôle prédominant dans la vasoconstriction pulmonaire, il semble donc essentiel de bloquer simultanément les récepteurs ETA et ETB afin d’inhiber la réponse vasoconstrictrice induite par l’ET. Mots-clés: endothéline-1, endothéline-3, artère de résistance pulmonaire, récepteur vasculaire, antagoniste des récepteurs à l’ET, dimérisation, phosphorothioate, hypertension artérielle pulmonaire

NMR studies of the conformational effect of single and double 3'-S-phosphorothiolate substitutions within deoxythymidine trinucleotides

NMR spectroscopy has been used to investigate the conformational effects of single and two consecutive 3′-S-phosphorothiolate modifications within a deoxythymidine trinucleotide. The presence of a single 3′-phosphorothioate modification shifts the conformation of the sugar ring it is attached to, from a mainly south to north pucker; this effect is also transmitted to the 3′-neighbour deoxyribose. This transmission is thought to be caused by favourable stacking of the heterocyclic bases. Similar observations have been made previously by this group. When two adjacent modifications are present, the conformations of the attached deoxyribose rings are again shifted almost completely to the north, however, there is no transmission to the 3′ deoxyribose ring. Base proton chemical shift analysis and molecular modelling have been used to aid elucidation of the origin of this feature. The observation for the dimodified sequence is consistent with our previously reported results for a related system in which spaced modifications are more thermodynamically stable than consecutive ones.