Modified dinucleoside tetraphosphonates, new potential inhibitors of HIV reverse transcriptase.

New gamma-substituted analogues of dNTP were synthesized and their enzymatic stability and antiviral properties were evaluated.

Anti-HIV activity of novel phosphonate derivatives of AZT, d4T, and ddA.

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.

Synthesis of [bis(inosine-5')]-tetraphosphate and [bis(inosine-5')]-pentaphosphate analogues bearing the residues of methylenediphosphonic acid.

Various methods of synthesis of metabolically stable phosphonate analogues of bisnucleoside oligophosphates containing two residues of methylenediphosphonic acid in the oligophosphate chain are studied. Phosphonate analogues of Ip4I and Ip5I are prepared.

Terminal deoxynucleotidyl transferase. catalysis of DNA (oligodeoxynucleotide) phosphorylation.

The phosphorylation and phosphonylation of the 3'-hydroxyl of oligodeoxynucleotide 3'-termini (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) are discussed. Palpha and Palpha, Pgamma-substituted modified triphosphates serve as low-molecular weight substrates in this reaction to give oligodeoxynucleotides with a 3'-phosphorylated or phosphonylated hydroxyl. The reaction is specific for TDT, and it is not catalyzed by avian myeloblastosis virus reverse transcriptase. The phosphate or phosphonate donor activities of modified triphosphates depend on their structure and increase with hydrophobicity. Several modified triphosphates demonstrated very high substrate activity, in some cases, up to one order of magnitude higher than that for dTTP. It has also been shown that TDT catalyzes primer extension with dinucleoside 5',5'-tetraphosphates as substrates.

Stereochemical control of DNA biosynthesis.

Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2'-deoxy-D-nucleotide residue by an enantiomeric L-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2'-deoxy-L-ribonucleoside 5'-triphosphates (L-dNTPs) as substrates. Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase. Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective. DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5',5'-tetraphosphates when L-dTTP was used as substrate. Comparison between models of template-primer complexes, modified or not by a single L-nucleotide residue, revealed striking differences in their geometry.

Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation.

The reaction of phosphorylation and phosphonylation of an oligodeoxynucleotide 3'-terminal hydroxyl (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) was found. Triphosphates modified at Palpha-, Palpha,gamma- or Palpha,beta,gamma-residues served as low-molecular weight substrates. The reaction was TDT specific; human DNA polymerasesalphaandbeta, as well as AMV reverse transcriptase did not catalyze it. The donor activity of modified triphosphates or triphosphonates depended on their structure and was increased with an increase in their hydrophobicity. The substrate activity of some modified triphosphates was up to one order of magnitude higher than that of ddTTP.

P-(alkyl)-nucleoside 5'-hydrogenphosphonates as depot forms of antiviral nucleotide analogues.

P-(Alkyl)esters of AZT 5'-hydrogenphosphonate were synthesized and their stabilities in the phosphate buffer and human serum were evaluated. The esters bearing residues of primary and secondary alcohols were degraded to give AZT, whereas those containing tertiary alkyl groups yielded AZT 5'-hydrogenphosphonate. The corresponding derivatives of d2A and d4T showed the same properties.

Reasons and limits of substrate activity of modified L-dNTP in DNA biosynthesis.

Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5'-triphosphates do not contain a substituent in position 3' DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3'-hydroxy group in L-dNTP prevents their incorporation into the DNA chain.

Antiviral activity and resistance profile of phosphazid--a novel prodrug of AZT.

Both AZT and its novel 5'-hydrogen phosphonate derivative, Phosphazid, possess similar in vitro activity and resistance profiles. Experiments involving AZT-resistant virus isolates revealed a strong correlation between resistance to AZT and cross-resistance to Phosphazid. In vitro selection for resistance to Phosphazid yielded viruses that were about 15-fold less sensitive than wild-type virus to this drug. Sequencing of the reverse transcriptase region of seven Phosphazid-selected viruses revealed a single codon mutation, D67N, that is associated with resistance to AZT.

Substrates of DNA polymerases with planar conformation of sugar: model of substrate transition state?

Several years ago, we published an hypothesis concerning conformation of the glycone moiety of different substrates in active centers of several DNA metabolizing enzymes (Nucleosides & Nucleotides 1993, 12, 649-670). This hypothesis prompted us to further study the subtle conformational changes on substrates of DNA polymerases. Data collected in our, as well as other laboratories, have been analyzed, and models of active centers of different DNA polymerases are discussed below. Based on the model of substrate requirements, we now can divide DNA polymerases into two distinguished classes.

2'-Deoxynucleoside 5'-triphosphates modified at alpha-, beta- and gamma-phosphates as substrates for DNA polymerases.

Replacement of alpha-, beta- and gamma-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at alpha-, beta- and gamma-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that gamma-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the gamma-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase alpha. For dUTP analogues substituted at both gamma-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at gamma-phosphonate or C5 position. Meanwhile, C5-substituted beta, gamma-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[beta, gamma-(methylphosphinyl)methylphosphonyl]-alpha-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.

Effect of triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity towards various DNA polymerases.

Some natural and glycon-modified dNTPs with beta,gamma-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases alpha and beta, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of beta,gamma-methylenediphosphonate substituted dTTP and 3'-azido-3'-deoxy-dTTP decreased in the following order: CF2 = CHF > CBr2 > CFMe >> CH2. This order is individual for each DNA polymerase. It is interesting to mention that beta,gamma-CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase beta. This specificity distinguishes DNA polymerase beta from other DNA polymerases studied.

Stereoisomers of deoxynucleoside 5'-triphosphates as substrates for template-dependent and -independent DNA polymerases.

All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.

Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase delta.

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

Conserved nucleotides of 23 S rRNA located at the ribosomal peptidyltransferase center.

Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schäfer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849-32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.

Should the asymmetry of enzymatic active centers always correlate with the asymmetry of their substrates?

Analysis of the substrate specificity of DNA polymerases and in part some other enzymes of nucleic acid metabolism towards unnatural L-stereomeric nucleosides and nucleotides was made. As a result, the hypothesis is proposed that when the chiral part of the substrate molecule does not interact specifically with the enzyme, substitution of natural D enantiomers by L compounds is possible.

Gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases.

Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.

Modified nucleotides as substrates and inhibitors of adenylate kinase from different sources.

The substrate and inhibitory properties of modified nucleotides with respect to adenylate kinase from rabbit muscles, human placenta and Escherichia coli were studied. A number of 5'-hydrogenphosphonates and 5'-fluorophosphates of modified nucleotides were shown to inhibit the phosphorylation reaction catalyzed by these enzymes. A clear difference between phosphonates of 3'-deoxyribonucleotides and the corresponding ribo- and 2',3'-dideoxyribonucleotides was found. 3'-Azido-2',3'-dideoxythymidine and its phosphorus derivatives did not inhibit the adenylate kinase reaction.