Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases β and λ

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) λ and β. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol λ; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol λ wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol β in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrate

Evaluation of the antiprotozoan properties of 5'-norcarbocyclic pyrimidine nucleosides

Carbocyclic nucleoside analogues have a distinguished history as anti-infectious agents, including key antiviral agents. Toxicity was initially a concern but this was reduced by the introduction of 5'-nor variants. Here, we report the result of our preliminary screening of a series of 5'-norcarbocyclic uridine analogues against protozoan parasites, specifically the major pathogens Leishmania mexicana and Trypanosoma brucei. The series displayed antiparasite activity in the low to mid-micromolar range and establishes a preliminary structure-activity relationship, with the 4',N(3)-di-(3,5-dimethylbenzoyl)-substituted analogues showing the most prominent activity. Utilizing an array of specially adapted cell lines, it was established that this series of analogues likely act through a common target. Moreover, the strong correlation between the trypanocidal and anti-leishmanial activities indicates that this mechanism is likely shared between the two species. EC50 values were unaffected by the disabling of pyrimidine biosynthesis in T. brucei, showing that these uridine analogues do not act directly on the enzymes of pyrimidine nucleotide metabolism. The lack of cross-resistance with 5-fluorouracil, also establishes that the carbocyclic analogues are not imported through the known uracil transporters, thus offering forth new insights for this class of nucleosides. The lack of cross-resistance with current trypanocides makes this compound class interesting for further exploration

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases β and λ

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) λ and β. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol λ; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol λ wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol β in a Mg(2+)-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates

5-arylaminouracil derivatives as potential dual-action agents

Several 5-aminouracil derivatives that have previously been shown to inhibit Mycobacterium tuberculosis growth at concentrations of 5-40 μg/mL are demonstrated to act also as noncompetitive non-nucleoside inhibitors of HIV-1 reverse transcriptase without causing toxicity in vitro (McyrillicT-4 cells) and ex vivo (human tonsillar tissue)

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 polymerase

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

Investigation of 5’-norcarbocyclic nucleoside analogues as antiprotozoal and antibacterial agents

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein

Organophosphorus S-adenosyl-L-methionine mimetics: synthesis, stability, and substrate properties

S-Adenosyl-l-methionine (SAM)-mediated methylation of biomolecules controls their function and regulates numerous vital intracellular processes. Analogs of SAM with a reporter group in place of the S-methyl group are widely used to study these processes. However, many of these analogs are chemically unstable that largely limits their practical application. We have developed a new compound, SAM-PH, which contains an H-phosphinic group (-P(O)(H)OH) instead of the SAM carboxylic group. SAM-PH is significantly more stable than SAM, retains functional activity in catechol-O-methyltransferase and methyltransferase WBSCR27 reactions. The last is associated with Williams–Beuren syndrome. Rac-SAM-PH was synthesized chemically, while (R,S)-SAM-PH and its analogs were prepared enzymatically either from H-phosphinic analogs of methionine (Met-PH) or H-phosphinic analog of S-adenosyl-l-homocysteine (SAH-PH) using methionine adenosyltransferase 2A or halide methyltransferases, respectively. SAH-PH undergoes glycoside bond cleavage in the presence of methylthioadenosine nucleosidase like natural SAH. Thus, SAM-PH and its analogs are promising new tools for investigating methyltransferases and incorporating reporter groups into their substrates