Chemical Architecture and Applications of Nucleic Acid Derivatives Containing 1,2,3-Triazole Functionalities Synthesized via Click Chemistry

There is considerable attention directed at chemically modifying nucleic acids with robust functional groups in order to alter their properties. Since the breakthrough of copper-assisted azide-alkyne cycloadditions (CuAAC), there have been several reports describing the synthesis and properties of novel triazole-modified nucleic acid derivatives for potential downstream DNA- and RNA-based applications. This review will focus on highlighting representative novel nucleic acid molecular structures that have been synthesized via the “click” azide-alkyne cycloaddition. Many of these derivatives show compatibility for various applications that involve enzymatic transformation, nucleic acid hybridization, molecular tagging and purification, and gene silencing. The details of these applications are discussed. In conclusion, the future of nucleic acid analogues functionalized with triazoles is promising

Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues

RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis

Plasmodium Purine Metabolism and Its Inhibition by Nucleoside and Nucleotide Analogues

International audienceMalaria still affects around 200 million people and is responsible for more than 400,000 deaths per year, mostly children in subequatorial areas. This disease is caused by parasites of the Plasmodium genus. Only a few WHO-recommended treatments are available to prevent or cure plasmodial infections, but genetic mutations in the causal parasites have led to onset of resistance against all commercial antimalarial drugs. New drugs and targets are being investigated to cope with this emerging problem, including enzymes belonging to the main metabolic pathways, while nucleoside and nucleotide analogues are also a promising class of potential drugs. This review highlights the main metabolic pathways targeted for the development of potential antiplasmodial therapies based on nucleos(t)ide analogues, as well as the different series of purine-containing nucleoside and nucleotide derivatives designed to inhibit Plasmodium falciparum purine metabolism.

Role of tautomerism in RNA biochemistry

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.National Institutes of Health (U.S.) (Grant P01 CA26731)National Institutes of Health (U.S.) (Grant R37 CA080024)National Institutes of Health (U.S.) (Grant P30 ES002109)National Institutes of Health (U.S.) (Training Grant T32 ES007020

Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (AT) and its photophysical characterization inside DNA. AT shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, AT shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that AT only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that AT shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, AT shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA

Mapping Drug Interactions at the Covalent Topoisomerase II-DNA Complex by Bisantrene/Amsacrine Congeners *

To identify structural determinants for the sequence-specific recognition of covalent topoisomerase II-DNA complexes by anti-cancer drugs, we investigated a number of bisantrene congeners, including a 10-azabioisoster, bearing one or two 4, 5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1, 4, or 9 of the anthracene ring system. The studied bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers were able to poison DNA topoisomerase II with an intermediate activity between those of bisantrene and m-AMSA. Moving the side chain from the central to a lateral ring (from C-9 to C-1/C-4) only slightly modified the drug DNA affinity, whereas it dramatically affected local base preferences of poison-stimulated DNA cleavage. In contrast, switching the planar aromatic systems of bisantrene and m-AMSA did not substantially alter the sequence specificity of drug action. A computer-assisted steric and electrostatic alignment analysis of the test compounds was in agreement with the experimental data, since a common pharmacophore was shared by bisantrene, m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of topoisomerase II poisons and in determining the poison pharmacophore

Exploiting nucleotide metabolism to improve cancer therapy : by targeting dUTPase, dCTPase and NUDT15

Synthetic nucleobase- and nucleoside-analogs have stood the test of time and remain a cornerstone in the treatment regimen against various forms of cancer. Due to their resemblance to endogenous nucleotides these antimetabolites interfere with cellular pathways, including nucleotide metabolism, as well as DNA and RNA synthesis. However, the treatment efficacy of nucleobase- and nucleoside-analogs can be hampered by “house-cleaning” enzymes involved in sanitation and balance of the nucleotide pool. In this work, we validated whether targeting nucleoside triphosphate hydrolases, involved in sanitation of the nucleotide pool, is a promising strategy to improve the efficacy of commonly used nucleobase- and nucleoside-analogs. These include: 1. dUTPase to potentiate 5-fluorouracil treatment 2. dCTPase to potentiate decitabine treatment 3. NUDT15 to potentiate 6-thioguanine treatment We characterized dUTPase, dCTPase and NUDT15 by various biochemical and biophysical techniques and assessed their role in intracellular nucleotide homeostasis using RNA interference. Through the development and use of small molecule inhibitors targeting these hydrolases, we highlighted the benefit of inhibiting nucleotide pool sanitization to improve nucleobase- and nucleoside-analog therapy. In Paper I we demonstrated that dUTPase inhibition reinforces 5-fluorouracil-induced replication defects and cytotoxicity. With this study, we contributed to the characterization of dUTPase inhibitors and increased our understanding of the mode of action of this combination treatment. In the second study (Paper II), we developed small molecule inhibitors against dCTPase to explore the biological function of this nucleoside triphosphate hydrolase in the context of endogenous nucleotide homeostasis and decitabine treatment. We showed that chronic inhibition of dCTPase has a cytostatic effect on cancer cells and potentiates cellular effects of decitabine therapy. Targeting enzymes involved in sanitation of the oxidized nucleotide pool is a novel treatment strategy that exploits the dysregulated reduction-oxidation environment of tumors. Based on the increasing attention to MTH1 as a prime example for this approach, we validated whether the sequence homolog NUDT15 (also known as MTH2) fulfills comparable enzymatic functions, making it a potential target for cancer therapy. With extensive biochemical and cellular experiments we demonstrated that NUDT15 possesses only minimal activity with oxidized nucleotides and is non-essential for cancer cell survival (Paper III). While assessing the cellular function of NUDT15, we discovered activity with the thiopurine effector metabolites, 6-thio-dGTP and 6-thio-GTP. In light of several pharmacogenetic studies, which link thiopurine hypersensitivity to the NUDT15 R139C variant, we further elucidated the role of NUDT15 (wild type and mutants) in thiopurine metabolism (Paper IV). We combined biochemical analyses with cellular experiments on genetically modified cell lines to demonstrate that NUDT15 has a strong preference for thiolated guanine substrates and that this activity counteracts thiopurine efficacy in cancer cells. Furthermore, we propose that the observed hypersensitivity of NUDT15 R139C positive patients is not caused by impaired enzymatic activity, but is a result ofuntenable protein stability. Inspired by these findings, we developed first-in-class NUDT15 inhibitors and validated whether pharmacological inhibition of NUDT15 is a promising strategy to sensitize leukemia cells to thiopurine treatment (Paper V). NUDT15 inhibition significantly increased the availability of thiopurine nucleoside triphosphates, leading to potentiation of DNA incorporation, DNA damage and cytotoxicity. Overall, these studies demonstrated that preventing the sanitation of nucleotide-analogs, by inhibiting nucleoside triphosphate hydrolases, is a promising strategy to improve the efficacy ofnucleobase- and nucleoside-analog treatments

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.DFG, 390540038, EXC 2008: UniSysCatDFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli