Photoreactive dTTP Analogues as Substrates for Thermostable DNA Polymerase from Thermus thermophilus B35

Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP–NAB-x-dUTP (where x = 2, 4, 7–13 is the number of atoms in the linker), were studied. All the analogues are substrates for thermostable Thermus thermophilus B35 DNA polymerase in the elongation reaction of the 5-³²P-labeled primer–template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUP residues into the 3 primer end did not impede further elongation of the chain in the presence of natural dNTP.     

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.     

Inhibitory effects of 5-alkyl- and 5-alkenyl-1-beta-D-arabinofuranosyluracil 5'-triphosphates on herpes virus-induced DNA polymerases

Various 5-substituted 1-beta-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and beta-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (microM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (microM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA polymerases, except for in the case of AraUTP itself.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligonucleotide analogues containing the internucleotide linker C3'-NH-CO-CH2-N(CH3)-C5'

Oligonucleotide analogues were synthesized whose internucleoside linker contains an amide bond and a methylamino group (C3'-NH-CO-CH2-N(CH3)-C5'). Melting curves for duplexes formed by modified oligonucleotides and natural oligonucleotides complementary to them were measured, and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified dinucleoside linker into the oligonucleotide only slightly decreases the melting temperatures of these duplexes compared with unmodified ones. The CD spectra of modified duplexes were studied, and their spatial structures are discussed.     

Comparative study of the efficacy of modifying DNA polymerase and DNA matrix by different photoactive groups at the 3'-end of the DNA primer

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoactivatable group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent binding of the [5'-32P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.     

Site-directed fragment-based generation of virtual sialic acid databases against influenza A hemagglutinin

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

Docking of sialic acid analogues against influenza A hemagglutinin: a correlational study between experimentally measured and computationally estimated affinities

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

Oligonucleotide analogues containing the internucleotide linker C3′-NH-CO-CH<Subscript>2</Subscript>-N(CH<Subscript>3</Subscript>)-C5′

Oligonucleotide analogues were synthesized whose internucleoside linker contains an amide bond and a methylamino group (C3′-NH-CO-CH₂-N(CH₃)-C5′). Melting curves for duplexes formed by modified oligonucleotides and natural oligonucleotides complementary to them were measured, and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified dinucleoside linker into the oligonucleotide only slightly decreases the melting temperatures of these duplexes compared with unmodified ones. The CD spectra of modified duplexes were studied, and their spatial structures are discussed.     

New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase]

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.     

Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.     

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.