Substrate/inhibition studies of bacteriophage T7 RNA polymerase with the 5′-triphosphate derivative of a ring-expanded (‘fat’) nucleoside possessing potent antiviral and anticancer activities

As part of an effort to explore the mechanism of potent, broad spectrum antiviral and anticancer activities of a number of ring-expanded (‘fat’) nucleosides that we recently reported, a representative ‘fat’ nucleoside 4,6-diamino-8-imino-8H-1-β- -ribofuranosylimidazo[4,5-e][1,3]diazepine (1) was converted to its 5′-triphosphate derivative (2), and biochemically screened for possible inhibition of nucleic acid polymerase activity, employing synthetic DNA templates and the bacteriophage T7 RNA polymerase as a representative polymerase. Our results suggest that 2 is a moderate inhibitor of T7 RNA polymerase, and that the 5′-triphosphate moiety of 2 appears to be essential for inhibition as nucleoside Scheme 1 and Scheme 2 alone failed to inhibit the polymerase reaction.

Scheme 2.     

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.     

Potent anti-hepatitis B viral activity and inhibition of bacteriophage T7 RNA polymerase by a "fat" nucleoside and its 5'-triphosphate derivative: synthetic, biochemical, and biological studies of 4,8-diamino-6-imino-6H-1-beta-D-ribofuranosyl-imidazo[4,5-E] [1,3]diazepine-5'-triphosphate.

The title nucleoside, 4,8-diamino-6-imino-6H-1-beta-d-ribofuranosylimidazo[4,5-e][1,3]-d iazepine, exhibited potent anti-hepatitis B viral activity with minimum toxicity in vitro, and its 5'-triphosphate derivative strongly inhibited the bacteriophage T7 RNA polymerase.     

Novel nucleoside triphosphate analogs for the enzymatic labeling of nucleic acids

We have evaluated several novel nucleotide analogs suitable for enzymatic labeling of nucleic acid targets for a variety of array-based assays. Two new reagents in particular, a C4-labeled 1-(2',3'-dideoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide 5'-triphosphate 5 and an N1-labeled 5-(beta-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5'-triphosphate 3, were found to be excellent substrates for labeling with terminal deoxynucleotidyl transferase and T7 RNA polymerase, respectively.     

Interaction of DNA polymerase I of Escherichia coli with nucleotides. Antagonistic effects of single-stranded polynucleotide homopolymers

Binding of deoxyribonucleoside 5'-triphosphates to DNA polymerase I of Escherichia coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5'-triphosphate/mol of DNA polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP greater than or equal to dATP greater than dCTP congruent to dTTP. These results are in agreement with previous findings [Englund, P. T., Huberman, J. A., Jovin, T. M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038-3044] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5'-triphosphates and nucleotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and non-complementary templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 +/- 10 monomer units of polynucleotides bound per molecule of DNA polymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5'-triphosphate into a rigid binding site.     

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.     

A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV). A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase(DV), ribavirin 5'-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 A. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.     

2'-Deoxy-2'-azidoadenosine triphosphate and 2'-deoxy-2'-fluoroadenosine triphosphate as substrates and inhibitors for Escherichia coli DNA-dependent RNA polymerase

The effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AZTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.     

Selective inhibition of initial polyadenylation in isolated nuclei by low levels of cordycepin 5"-triphosphate.

The effect of cordycepin 5'-triphosphate on poly(A) synthesis was investigated in isolated rat hepatic nuclei. Nuclei were incubated in the absence and presence of exogenous primer in order to distinguish the chromatin-associated poly(A) polymerase from the "free" enzyme (Jacob, S.T., Roe, F.J. and Rose, K.M. (1976) Biochem. J. 153, 733--735). The chromatin-bound enzyme, which adds adenylate residues onto the endogenous RNA, was selectively inhibited at low concentrations of cordycepin 5'-triphosphate, 50% inhibition being achieved at 2microng/ml. At least 80 times more inhibitor was required for 50% reduction in the "free" nuclear poly(A) polymerase activity. Inhibition of DNA-dependent RNA synthesis also required higher concentrations of the nucleotide analogue. These data not only offer a mechanism for the selective inhibition of initial polyadenylation of heterogeneous nuclear RNA in vivo by cordycepin, but also provide a satisfactory explanation for the indiscriminate effect of the inhibitor on partially purified or "free" poly(A) and RNA polymerases.     

Inhibition of replication of human hepatitis B virus

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.     

The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.