Deoxythioguanosine triphosphate impairs HIV replication: a new mechanism for an old drug.

Inhibition of HIV-1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti-retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV-1 RT and then to abrogate RNA cleavage by HIV-1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC(50)=0.035 microM, IC(95)=15.4 microM), with minimal toxicity to host lymphocytes (<10% at 15.4 microM TG, P<0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume=19 microM(2)%), without additive cytotoxicity to host lymphocytes. Thus, thiopurines are novel anti-retroviral agents that alter the DNA-RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.

A novel protein complex distinct from mismatch repair binds thioguanylated DNA.

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSalpha) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSalpha complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.

Thioguanine substitution alters DNA cleavage mediated by topoisomerase II.

Thiopurines and topoisomerase II-targeted drugs (e.g., etoposide) are widely used anticancer drugs. However, topoisomerase II-targeted drugs can cause acute myeloid leukemia, with the risk of this secondary leukemia linked to a genetic defect in thiopurine catabolism. Chronic thiopurines result in thioguanine substitution in DNA. The effect of these substitutions on DNA topoisomerase II activity is not known. Our goal was to determine whether deoxythioguanosine substitution alters DNA cleavage stabilized by human topoisomerase II. We studied four variations of a 40 mer oligonucleotide with a topoisomerase II cleavage site, each with a single deoxythioguanosine in a different position relative to the cleavage site (-1 or +2 in the top and +2 or +4 in the bottom strand). Deoxythioguanosine substitution caused position-dependent quantitative effects on cleavage. With the -1 or +2 top and +2 or +4 bottom substitutions, mean topoisomerase II-induced cleavage was 0.6-, 2.0-, 1.1-, and 3.3-fold that with the wild-type substrate (P=0. 011, < 0.008, 0.51, and < 0.001, respectively). In the presence of 100 microM etoposide, cleavage was enhanced for wild-type and all thioguanosine-modified substrates relative to no etoposide, with the +4 bottom substitution showing greater etoposide-induced cleavage than the wild-type substrate (P=0.015). We conclude that thioguanine incorporation alters the DNA cleavage induced by topoisomerase II in the presence and absence of etoposide, providing new insights to the mechanism of thiopurine effect and on the leukemogenesis of thiopurines, with or without topoisomerase inhibitors.

Human RNase H-mediated RNA cleavage from DNA-RNA duplexes is inhibited by 6-deoxythioguanosine incorporation into DNA.

Mercaptopurine and thioguanine are anticancer and immunosuppressive agents that exert their primary cytotoxic effects via incorporation of deoxythioguanosine (dG(s)) into DNA, but the precise mechanism(s) by which this causes cytotoxicity remains unknown. We initially determined that the level of dG(s) incorporation into DNA of human T- and B-lineage leukemia cell lines did not correlate significantly with the extent of cytotoxicity (IC(50)), except that there was no cytotoxicity in the absence of dG(s) incorporation. To elucidate biological processes perturbed by dG(s) incorporation into DNA, we chemically synthesized oligodeoxyribonucleotides containing a single dG(s) (11 mer and 19 mer), which decreased the melting temperature (T(m)) of DNA-DNA duplexes without major structural changes, as evidenced by circular dichroism spectra. Using nuclear extracts from human lymphoblastic leukemia cells (CCRF-CEM, NALM6, and Molt4), we documented that dG(s) incorporation into the DNA strand of DNA-RNA heteroduplexes significantly inhibited human RNase H-catalyzed RNA cleavage (80-90% inhibition) and that a similar inhibition was evident with bacterial RNase H. These data provide the first evidence that thiopurines inhibit the function of RNase H, indicating that their mechanism of cytotoxicity may involve interference with this component of the replication machinery.

Synthesis of modified thiopurine nucleosides for structural characterization of human thiopurine S-methyltransferase.

Synthesis of a number of photoactive thiopurine-containing nucleosides was described. S-methylation of the synthesized compounds in the course of the reaction catalyzed by recombinant human thiopurine S-methyltransferase was studied by UV-spectroscopy.

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.

Methylation of mercaptopurine, thioguanine, and their nucleotide metabolites by heterologously expressed human thiopurine S-methyltransferase.

Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, catalyzes S-methylation of mercaptopurine (MP) and thioguanine (TG), yielding S-methylated nucleobases that are inactive, whereas S-methylated nucleotides of these thiopurines are cytotoxic. A yeast-based heterologous expression system was therefore used to characterize human TPMT-catalyzed methylation of MP, TG, and their principal nucleotide metabolites [thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively]. MP, TG, TIMP, and TGMP were all substrates for human TPMT, exhibiting similar Michaelis-Menten kinetic parameters (Km, 10.6-27.1 microM; Vmax, 31-59 nmol/min/mg of TPMT). Consistent with these kinetic parameters, human leukemia cells (CEM) incubated for 24 hr with 10 microM MP or TG accumulated significantly higher (2.3-fold, p = 0.01) concentrations of methyl-TIMP after MP incubation than methyl-TGMP after TG incubation, due to the 2.7-fold higher concentration of TIMP after MP incubation, compared with TG nucleotides (TGN) after TG incubation. Moreover, intracellular accumulation of TGN was 2.5-fold greater after TG incubation than after MP incubation (p = 0.01). These data establish that MP, TG, and their principal nucleotide metabolites are comparable substrates for polymorphic TPMT, and they demonstrate significant differences in the accumulation of active TGN and methylated nucleotides when leukemia cells are treated with MP versus TG.

[Cobalt-corrin oligonucleotide derivatives as reagents for selective cleavage of nucleic acids]. / Kobal't-korrinovye proizvodnye oligonukleotidov kak reagenty dlia izbiratel'nogo rasshepleniia nukleinovykh kislot.

Techniques for preparing catalytically active corrin complexes of cobalt with monofunctional e-carboxyl- and e-amino groups in a macroligand are suggested. Two methods of the covalent attachment of such Co-corrin complexes to the terminal 3'- and 5'-phosphate groups of oligodeoxyribonucleotides were studied: the introduction of the complex to the oligonucleotide after oligonucleotide synthesis in aqueous solution and in the course of automated solid-phase oligonucleotide synthesis. Introducing of the Co-corrin complex during solid-phase synthesis was more efficient. It was demonstrated that the oligonucleotide probe thus obtained was able to act as a nuclease in the presence of ascorbic acid, the location of the clearage sites being determined by the addressing oligonucleotide.

[Interaction of RNAase H from E. coli with modified hybrid duplexes. I. Duplexes modified at the carbohydrate residue]. / Vzaimodeistvie RNKazy H iz E. coli s modifitsirovannymi gibridnymi dupleksami. I. Dupleksy, soderzhashchie modifikatsii po uglevodnomu ostatku.

The influence of oligodeoxyribonucleotide probes containing 1-(D-beta-2'-deoxythreo-pentofuranosyl)thymine or 1-(D-beta-2'-deoxy-2'-fluoro-pentofuranosyl)uracil on the ability of the hybrid duplexes to interact with RNase H from E. coli was studied. A kinetic approach was used to measure of the modification effect. The hybrid duplex, prA18/d(TTflU)6TT, was shown not to interact with RNase H, whereas prA18/d(xTTT)6 inhibited the RNase H activity (Ki = 0.67 mkM). The thermostability of the modified duplexes was estimated. The present technique may lead to the use of some modified oligonucleotides as antisences.

[Antisense oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine at the 3'- and 5'-end]. / Antisensovye oligonukleotidy, soderzhashchie po 3'- i 5'-kontsam 1-(beta-D'-dezoksi-treo-pentofuranozil)timin.

Oligonucleotides with 3'- and 5'-terminal 1-(beta-D-2'-deoxy-threo-pentafuranosyl)thymin residues (inversion at C3'-atom) were obtained by the automatic phosphoramidite synthesis. The modifications protect oligonucleotides from the nuclease degradation and do not affect the stability of nucleic acid duplexes.

[Synthesis of cyclic oligodeoxyribonucleotides with non-nucleotide inserts]. / Sintez tsiklicheskikh oligodezoksiribonukleotidov s nenukleotidnymi vstavkami.

An effective method for the oligonucleotide cyclization using BrCN-induced chemical ligation was developed. The novel idea to incorporate non-nucleotide inserts makes the process of cyclizations independent of the inner secondary structure of the linear precursor. An set of assays were developed to confirm the cyclic structure of the compounds obtained.

[Oligodeoxyribonucleotides containing 2'-amino-2'-deoxypyrimidine nucleosides]. / Oligodezoksiribonukleotidy, soderzhashchie 2'-amino-2'-dezoksipirimidinovye nukleozidy.

The synthesis, by means of the standard phosphoramidite chemistry, of modified oligodeoxynucleotides (5-20 residues) containing 2'-amino-2'-deoxypyrimidine nucleosides has been carried out, and their ability to form duplexes with complementary DNA has been investigated. A high reactivity of such compounds in N-acylation was shown.

[Hybridase cleavage of RNA. V. Complementary precision of cleavage]. / Gibridaznoe rasshcheplenie RNK. V. Komplementatsionnaia tochnost' rasshchepleniia.

The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.

[Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]. / Gibridaznoe rasshcheplenie RNK. IV. Oligonukleotidnye zondy, soderzhashchie 2'-dezoksi-2'-ftornukleozidy i arabinofuranoziltsitozin.

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.

[Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties]. / Gibridaznoe rasshcheplenie RNK. III. Ispol'zovanie UF-spektroskopii dlia izucheniia kineticheskikh svoistv RNKazy N iz E. coli.

A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.

[Hybridase cleavage of RNA. II. Automatic synthesis of mixed oligonucleotide probes]. / Gibridaznoe rasshcheplenie RNK. II. Avtomaticheskii sintez smeshannykh oligonukleotidnykh zondov.

Several oligo(ribodeoxyribo)nucleotides to be used as probes in the RNase H-induced hydrolyses of 5S ribosomal RNA E. coli were synthesized on the Victoria-4M automatic gene synthesizer by the phosphoramidite approach, which allows for introducing ribonucleotides into any position of the oligomer. 2'-Hydroxy function was protected by tert-butyldimethylsilyl group whose hydrophobicity simplified isolation of the oligonucleotides by reverse-phased HPLC.

[Hybridase cleavage of RNA. I. Oligonucleotide probes for regiospecific RNA cleavage]. / Gibridaznoe rasshcheplenie RNK. I. Oligonukleotidnye zondy dlia regiospetsificheskogo rasshchepleniia RNK.

New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.

[Increased selectivity of directed RNA hydrolysis mediated by RNAse H from E. coli using mixed oligo(deoxyribo-ribo)nucleotides]. / Povyshenie izbiratel'nosti napravlennogo gidroliza RNK RNKazoi H iz Ecoli s pomoshch'iu smeshannykh oligo(dezoksiribo-ribo)-nukleotidov.

Chimeric oligo(deoxyribo-ribo)nucleotides appeared to be a valuable tool to achieve the high selectivity of RNA cleavage as shown by RNA-ase H-mediated hydrolysis of TMV RNA directed by d(TGTGTATGCC), d(TGTGTAT), d(TGTGTAT)GCCAU and d(TGTGTAT)ppGCCAU.