Lipophilic pro-oligonucleotides are rapidly and efficiently internalized in HeLa cells.

Model t-Bu-SATE pro-dodecathymidines labeled with fluorescein and exhibiting various lipophilicities were evaluated for their uptake by cells in culture. Pro-oligonucleotides with appropriate lipophilicity were found to permeate across the HeLa cell membrane much more extensively than the control phosphorothioate oligo or than the hydrophilic pro-oligos. Fluorescence patterns of internalization were consistent with a diffusion mechanism resulting in the appearance of a uniform cytoplasmic distribution and nuclear accumulation, as confirmed by confocal microscopy.

Aerobic and hypoxic toxicity of a new class of mixed-function drugs associating nitroimidazoles and chloroethylnitrosourea in nitrosourea-sensitive (Mer-) and -resistant (Mer+) human tumor cells.

The cytotoxicity of two series (A and B) of novel mixed-function compounds (NI-CENU) combining nitroimidazole (NI) and chloroethylnitrosourea (CENU) functions were examined in Mer- HeLa-MR and Mer+ HeLa-S3 cells. Series A compounds differed from those in Series B by having a hydroxypropyl as opposed to an ethyl group linking the imidazole ring and the nitrosoureido function. Four analogues, including the imidazole and the 2-, 4-, and 5-NO2 derivatives, were evaluated in each series. Cells were exposed to the various compounds for 4 h under aerobic and hypoxic conditions, and toxicity was assessed by clonogenic assay. Corresponding analogues in Series A and B were equally toxic to HeLa-MR cells. Preferential hypoxic toxicity was observed only with the 2-NO2 derivative in either series (I-278, Series A; I-282, Series B). For either compound a dose enhancement factor of 2.4 was observed for hypoxic exposures. The Mer+ HeLa-S3 cells were considerably more resistant to the NI-CENU than were their HeLa-MR counterparts. In further contrast to the HeLa-MR data, the Series B compounds were consistently more effective against the HeLa-S3 cells than were their corresponding Series A analogues. The enhanced effectiveness of the Series B compounds in HeLa-S3 cells may be related to the fact that these compounds express carbamoylating activity whereas Series A compounds lack this property. Again only I-278 and I-282 were preferentially toxic to hypoxic cells; however, the aerobic/hypoxic differential was dramatically reduced (dose enhancement factor = 1.3) as compared to that observed with the HeLa-MR cells. The enhanced hypoxic toxicity of the 2-NO2 NI-CENUs was not due to direct hypoxic toxicity of the nitro moiety but presumably is the result of enhancement of CENU toxicity (i.e., chemosensitization). The data suggest that much lower concentrations of NI may be required to observe chemosensitization when the NI and chemotherapeutic agent are administered as a single mixed-function compound.

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.

Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.

Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.

Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase.

Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A):poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself. Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Antiviral activity of conjugates between poly(L-lysine) and synthetic oligodeoxyribonucleotides.

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell internalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (beta)-anomeric oligos to poly(L-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 microM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences, i.e., to untranscribed regions. No antiviral activity can be obtained with (alpha)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.

Degradation by the 2',5'-phosphodiesterase activity of mouse cells requires the presence of a ribo hydroxyl group in the penultimate position of the oligonucleotide substrate.

A series of 9-beta-D-xylofuranosyladenine (xyloA or xyloadenosine) substituted analogs of 2-5A core trimer and tetramer were examined for their ability to be degraded by the 2',5'-phosphodiesterase activity of cytoplasmic extracts of mouse L cells. Two distinct groups of xyloA-substituted analogs could be readily discriminated. The first group contained xyloadenosine at the 2'-termini and included A2'p5'A2'p5'(xyloA) and A2'p5'A2'p5'A2'p5'(xyloA). These oligomers behaved as did their parent oligoadenylates in that they were equally sensitive to degradation by the 2',5'-phosphodiesterase activity. The second group of oligonucleotides bore a xyloadenosine residue in the penultimate nucleotide residues of the oligomers and included A2'p5'(xyloA)2'p5'(xyloA), (xyloA)2'p5'(xyloA)2'p5'(xyloA), A2'p5'A2'p5'(xyloA)2'p5'(xyloA) and (xyloA)2'p5' (xyloA)2'p5'(xyloA)2'p5'(xyloA). This group was quite resistant to 2',5'-phosphodiesterase activity. In all, the findings demonstrate that the ribo configuration 3'-hydroxyl group in the penultimate nucleotide of the oligonucleotide substrate is a prerequisite for the 2',5'-phosphodiesterase activity.

Chimeric alpha-beta oligonucleotides as antisense inhibitors of reverse transcription.

Alpha-beta chimeric 17-mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha-beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell-free system to various extent, via the degradation of the RNA target by RNase H.

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs.

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.

Synthesis and antitumor evaluation of some nitrosourea and nitrogen mustard amino acid derivatives.

A series of (2-chloroethyl)nitrosourea and nitrogen mustard amino acid derivatives have been synthesized for antitumor evaluation. Reaction of an appropriate N-protected amino acid with 2-chloroethylamine followed by removal of the N-protecting group and condensation with an active (2-chloroethyl)nitrosocarbamate yielded N-[(2-chloroethyl)nitrosocarbamoyl]amino acid (2-chloroethyl)amides. Antitumor evaluation was performed against leukemia L1210, in vivo, in mice. These derivatives exhibited very interesting activities, particularly the sarcosine and gamma-aminobutyric acid derivatives.

Activated N-nitrosocarbamates for regioselective synthesis of N-nitrosoureas.

A practical and convenient method for synthesizing antitumor compounds, N-alkyl-N-nitrosoureas, regioselectively nitrosated on the nitrogen atom bearing the alkyl group is proposed. N-Alkyl-N-nitrosocarbamates are interesting intermediates in these syntheses and yield, by reaction with amino compounds, the regioselectively nitrosated N-alkyl-N-nitrosoureas. As an interesting example, N,N'-bis[(2-chloroethyl)nitrosocarbamoyl]cystamine, a new attractive oncostatic derivative, has been prepared. The cytotoxic activity of these various compounds were tested on L1210 leukemia.

Systematic synthesis and biological evaluation of alpha- and beta-D-lyxofuranosyl nucleosides of the five naturally occurring nucleic acid bases.

The alpha- and beta-D-lyxofuranosyl analogues of the naturally occurring nucleosides have been synthesized and their antiviral properties examined. The alpha anomers were prepared by glycosylation of purine and pyrimidine aglycons with tetra-O-acetyl-alpha-D-lyxofuranose, followed by removal of the blocking groups. The beta anomers were obtained by sequential oxidation and reduction of 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-beta-D-x ylofuranosyl nucleosides. The lyxofuranosyl nucleosides were tested for their activity against a variety of RNA and DNA viruses and for inhibition of cell growth. One compound, 9-alpha-D-lyxofuranosyladenine, showed activity against herpes simplex virus types 1 and 2 both in vitro and in vivo.

Systematic synthesis and biological evaluation of alpha- and beta-D-xylofuranosyl nucleosides of the five naturally occurring bases in nucleic acids and related analogues.

The alpha- and beta-D-xylofuranosyl analogues of the naturally occurring nucleosides, as well as other D-xylofuranosyl derivatives, have been the subject of a systematic synthesis and examination of their biological, i.e. antiviral antimetabolic, and cytostatic properties. The beta anomers were prepared by glycosylation of purine and pyrimidine aglycons with peracylated 1-O-acetyl-alpha-D-xylofuranoses, followed by removal of the blocking groups. The alpha anomers were obtained by a multistep synthesis with use of 2-amino- or 2-mercapto-alpha-D-xylofuran[1',2':4,5]-2-oxazoline as starting material. The xylofuranosyl nucleosides were tested for their activity against a variety of RNA and DNA viruses and for inhibition of cell growth and macromolecule synthesis. Three compounds, 9-(beta-D-xylofuranosyl)adenine, 9-(beta-D-xylofuranosyl)guanine, and 1-(beta-D-xylofuranosyl)cytosine, showed marked biological activity.

Synthesis and biological evaluation of isomeric dinucleoside monophosphates and monomethylphosphonates of 9-beta-D-arabinofuranosyladenine and related analogues.

The 3'----5',3'----3' and 5'----5' dinucleoside monophosphates and methylphosphonates of 9-beta-D-arabinofuranosyladenine, as well as its 5'-(hydrogen phosphonate) and 5'-(methyl methylphosphonate) derivatives have been the subject of a systematic synthesis and examination of their biological, i.e. antiviral and cytostatic, properties. First the properly protected monomeric building blocks were prepared and then condensed to give fully protected intermediates. These latter were then deblocked to afford the unprotected compounds, which were fully characterized. Only the 3'----5' phosphodiester isomers 13 and 16 and, to a lesser extent, the 5'-(hydrogen phosphonate) derivative 21 showed marked biological activity.

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine.

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.

S-Acyl-2-thioethyl aryl phosphotriester derivatives as mononucleotide prodrugs.

The synthesis and biological activities of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a phenyl group or L-tyrosinyl residues are reported. The target compounds were obtained via either P(V) or P(III) chemistry from the appropriate aryl precursors. All the derivatives were evaluated for their in vitro anti-HIV activity, and they appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments, with EC(50) values between the micro- and nanomolar range. Furthermore, compounds incorporating an amino- and/or acid-substituted tyrosinyl residue demonstrated significant anti-HIV effects in thymidine kinase-deficient (TK(-)) cells showing their ability to act as mononucleotide prodrugs. The proposed decomposition process of these mixed mononucleoside aryl phosphotriesters may involve esterase activation followed by phosphodiesterase hydrolysis.

Synthesis and radioprotective activity of dipeptide cysteamine and cystamine derivatives.

Some N-(dipeptidyl)-S-acetylcysteamine and N,N'-(dipeptidyl)cystamine salt derivatives were synthesized and evaluated as candidate radioprotector agents. Toxicity and radioprotective activity as the dose reduction factor (DRF) were determined in vivo on mice and compared to N-glycyl-S-acetylcysteamine trifluoroacetate. One of the most interesting compounds of this series was N-glycylglycyl-S-acetylcysteamine trifluoroacetate (8).

Acyclic analogues of 3'-azido-3'-deoxythymidine as potential antiviral agents. Nucleoside synthesis by Michael addition.

An acyclic derivative of 3'-azido-3'-deoxythymidine, (R,S)-1-[1-(2-hydroxyethoxy)-3-azidopropyl]thymine (2), has been synthesized by a path involving Michael-type addition. Related thymidine analogues lacking the C(3')-C(4') bond were similarly obtained. The method provides a versatile new route to nucleoside analogues. The new compounds were found to be essentially inactive against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro when tested up to 100 microM.

Synthesis and radioprotective activity of new cysteamine and cystamine derivatives.

A variety of N-(aminoalkanoyl)-S-acylcysteamine and N,N'-bis(aminoalkanoyl)cystamine salt derivatives were synthesized. Toxicity and radioprotective activity (as the dose reduction factor DRF) were determined in vivo on mice and compared to WR 2721 and S-acetylcysteamine hydrochloride. One of the most interesting compounds of this series was N-glycyl-S-acetylcysteamine trifluoroacetate (16, I 102). Structure-activity relationships are discussed.

Comparative evaluation of seven oligonucleotide analogues as potential antisense agents.

12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).