Maxim V. Jasko

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 δ (pol δ) 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 δ on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol δ 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 δ, the ability to incorporate (and/or not excise) the dTTP analog, 2′-deoxythymidine-5′-O-(α-phosphonomethyl)-β,γ-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol δtemplate-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol δ. This model suggests an explicit mechanistic requirement for the intrinsic 3′-5′-exonuclease of pol δ.

Synthesis and antiherpetic activity of acyclovir phosphonates.

Abstract Phosphonate derivatives of acyclovir containing phosphorous acid and ethoxycarbonylphosphonic acid residues as well as their isopropyl esters were prepared. They selectively inhibited the herpes simplex virus 1 reproduction in Vero cell culture, the efficacy of esters being 3–4 times higher than that of ACV. The hydrolysis of the synthesized compounds was studied in the PBS buffer and human blood serum.

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

Some natural and glycon‐modified dNTPs with β,γ‐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 α and β, 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 β,γ‐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 β,γ‐CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase β. This specificity distinguishes DNA polymerase β from other DNA polymerases studied.

5′-Aminocarbonyl Phosphonates as New Zidovudine Depot Forms: Antiviral Properties, Intracellular Transformations, and Pharmacokinetic Parameters

The main disadvantages of 3′-azido-3′-deoxythymidine (zidovudine, AZT), the most common anti-HIV drug, are toxicity and a short half-life in the organism. The introduction of an H-phosphonate group into the AZT 5′ position resulted in significant improvement of its therapeutic properties and allowed a new anti-HIV drug, Nikavir (AZT H-phosphonate). In this work, we described a new group of AZT derivatives, namely, AZT 5′-aminocarbonylphosphonates. The synthesized compounds displayed antiviral properties in cell cultures infected with HIV-1 and the capacity to release the active nucleoside in animals (rabbits and dogs) in a dose-dependent manner. The compounds were less toxic in MT-4 and HL-60 cell cultures and experimental animals compared with AZT. Major metabolites found in MT-4 cells after their incubation with AZT 5′-aminocarbonylphosphonate 1 were AZT and AZT 5′-phosphate (25 and 55%, respectively). Among the tested compounds, phosphonate 1 was the most effective AZT donor, and its longest t1/2 and Tmax values in the line phosphonate 1 - AZT H-phosphonate - AZT imply that compound 1 is an extended depot form of AZT. Although bioavailability of AZT after oral administration of phosphonate 1 was lower than those of AZT H-phosphonate and AZT (8 against 14 and 49%), we expect that this reduction would not cause essential decrease of antiviral activity but noticeably decrease toxicity as a result of gradual accumulation of AZT in blood and the absence of sharp difference between Cmax and Cmin. Such a combination of properties makes the compounds of this group promising for further studies as extended-release forms of AZT.

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases β and λ

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the α-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases α, β and λ, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase β incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase λ could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase λ Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.

5′-Phosphonates of Ribonucleosides and 2′-Deoxyribonucleosides: Synthesis and Antiviral Activity

Abstract 5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several...

Synthesis of novel thymidine derivatives containing a polycyclic tetrazole linker

Abstract The synthesis of mono- and bis-3′-substituted thymidine derivatives with a polycyclic tetrazole linker (1,5-bis(tetrazol-5-yl)-3-oxapentane) is described.

Phosphoramidate derivatives of acyclovir: synthesis and antiviral activity in HIV-1 and HSV-1 models in vitro.

The antiviral activity against HIV and HSV and the chemical stability of ACV phosphoramidate derivatives were studied. The phosphoramidates of ACV demonstrated moderate activity. The best compound appeared to be 9-(2-hydroxymethyl)guanine phosphoromonomorpholidate (7), which inhibited virus replication in pseudo-HIV-1 particles by 50% at 50 μM. It also inhibited replication of wild-type HSV-1 (9.7 μM) as well as an acyclovir-resistant strain (25 μM). None of the synthesised compounds showed any cytotoxicity.

Cell Metabolism of Acyclovir Phosphonate Derivatives and Antiherpesvirus Activity of their Combinations with α2-Interferon

The combinational use of acyclovir (ACV) phosphonate esters and α2‐interferon was shown to produce a synergistic effect on inhibition of HSV‐1 replication in Vero cell cultures. Unlike other acyclovir phosphonate derivatives studied earlier, ACV H‐phosphonate is not an ACV prodrug. On penetrating into the cells, it may be directly converted into ACV monophosphate escaping dephosphonylation–phosphorylation steps.

dNTP modified at triphosphate residues: substrate properties towards DNA polymerases and stability in human serum.

Substrate and terminating substrate properties of dNTP with phosphate groups replaced by phosphonates at alpha-, gamma-, beta, gamma-, and alpha, beta, gamma-positions towards different human DNA polymerases and retroviral reverse transcriptases are reviewed. Substitution of the phosphate group by the phosphonate at any of the three phosphate positions of dNTP increased their stability towards dephosphorylating enzymes of human blood. In some cases hydrophobicity of these compounds was markedly enhanced.