Inhibiting DNA synthesis is an important therapeutic strategy that is widely used to treat a number of hyperproliferative diseases including viral infections, autoimmune disorders, and cancer. This chapter describes two major categories of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrmidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents including cisplatin and chlorambucil that modify the composition and structure of the nucleic acid substrate to indirectly inhibit DNA synthesis. Special emphasis is placed on describing the molecular mechanisms of these inhibitory effects against chromosomal and mitochondrial DNA polymerases. Discussions are also provided on the mechanisms associated with resistance to these therapeutic agents. A primary focus is toward understanding the roles of specialized DNA polymerases that by-pass DNA lesions produced by DNA damaging agents. Finally, a section is provided that describes emerging areas in developing new therapeutic strategies targeting specialized DNA polymerases.

DNA is often referred to as the “molecule of life” since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, recent chemical and biochemical studies using nucleobase-modified nucleotides that contain “non-hydrogen bonding” functional groups have challenged many of the dogmatic views for the necessity of hydrogen-bonding interactions for DNA stability and function. Based on years of exciting research, this area has expanded tremendously and is thus too expansive to provide a comprehensive review on the topic. As such, this review article provides an opinion highlighting how nucleobase-modified nucleotides are being applied in diverse biomedical fields, focusing on three exciting areas of research. The first section addresses how these analogs are used as mechanistic probes for DNA polymerase activity and fidelity during replication. This section outlines the synthetic logic and medicinal chemistry approaches used to replace hydrogen-bonding functional groups to examine the contributions of shape/size, nucleobase hydrophobicity, and pi-electron interactions. The second section extends these mechanistic studies to provide insight into how nucleobase-modified nucleosides are used in synthetic biology. One example is through expansion of the genetic code in which changing the composition of DNA makes it possible to site-specifically incorporate unnatural amino acids bearing unique functional groups into enzymes and receptors. The final section describes results of pre-clinical studies using nucleobase-modified nucleosides as potential therapeutic agents against diseases such as cancer.

DNA polymerases catalyze the insertion of a nucleoside triphosphate into the growing polymer chain using the template strand as a guide. Numerous factors such as hydrogen bonding interactions, base-stacking contributions, and desolvation play important roles in controlling the efficiency and fidelity of this process. We previously demonstrated that 5-nitro-indolyl-2′-deoxyriboside triphosphate, a non-natural nucleobase with enhanced base-stacking properties, was more efficiently inserted opposite a non-templating DNA lesion compared to natural templating nucleobases (E. Z. Reineks and A. J. Berdis, Biochemistry, 2004, 43, 393–404). The catalytic enhancement was proposed to reflect increased base-stacking interactions of the non-natural nucleobase with the polymerase and DNA. However, the effects of desolvation could not be unambiguously refuted. To further address the contributions of base stacking and desolvation during translesion DNA replication, we synthesized indolyl-2′-deoxyriboside triphosphate, a nucleobase devoid of nitro groups, and measured its efficiency of enzymatic insertion into modified and unmodified DNA. Removal of the nitro group reduces the catalytic efficiency for insertion opposite an abasic site by 3600-fold. This results from a large decrease in the rate of polymerization (∼450-fold) coupled with a modest decrease in binding affinity (∼8-fold). Since both non-natural nucleobases show the same degree of hydrophobicity, we attribute this reduction to the loss of base-stacking contributions rather than desolvation capabilities. Indolyl-2′-deoxyriboside triphosphate can also be inserted opposite natural nucleobases. Surprisingly, the catalytic efficiency for insertion is nearly identical to that measured for insertion opposite an abasic site. These data are discussed within the context of π-electron interactions of the incoming nucleobase with the polymerase:DNA complex. Despite this lack of insertion selectivity, the polymerase is unable to extend beyond the non-natural nucleobase. This result indicates that indolyl-2′-deoxyriboside triphosphate acts as an indiscriminate chain terminator of DNA synthesis that may have unique therapeutic applications.

Temozolomide is a DNA-alkylating agent used to treat brain tumors, but resistance to this drug is common. In this study, we provide evidence that efficacious responses to this drug can be heightened significantly by coadministration of an artificial nucleoside (5-nitroindolyl-2′-deoxyriboside, 5-NIdR) that efficiently and selectively inhibits the replication of DNA lesions generated by temozolomide. Conversion of this compound to the corresponding nucleoside triphosphate, 5-nitroindolyl-2′-deoxyriboside triphosphate, in vivo creates a potent inhibitor of several human DNA polymerases that can replicate damaged DNA. Accordingly, 5-NIdR synergized with temozolomide to increase apoptosis of tumor cells. In a murine xenograft model of glioblastoma, whereas temozolomide only delayed tumor growth, its coadministration with 5-NIdR caused complete tumor regression. Exploratory toxicology investigations showed that high doses of 5-NIdR did not produce the side effects commonly seen with conventional nucleoside analogs. Collectively, our results offer a preclinical pharmacologic proof of concept for the coordinate inhibition of translesion DNA synthesis as a strategy to improve chemotherapeutic responses in aggressive brain tumors. Significance: Combinatorial treatment of glioblastoma with temozolomide and a novel artificial nucleoside that inhibits replication of damaged DNA can safely enhance therapeutic responses. Cancer Res; 78(4); 1083–96. ©2017 AACR.

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the π-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and π-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2′-deoxyriboside 5′-triphosphate (5-CyITP), 5-ethyleneindolyl-2′-deoxyriboside 5′-triphosphate (5-EyITP), 5-methylindolyl-2′-deoxyriboside 5′-triphosphate (5-MeITP), and 5-ethylindolyl-2′-deoxyriboside 5′-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall π-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA ∼100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.