Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer

Abstract

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.

Keywords: DNA damaging agents; DNA polymerases; cancer; chemotherapy; nucleoside analogs.

Figure 1

DNA polymerases use a common mechanism to synthesize DNA. During the polymerization process, a nucleotide is covalently attached to the 3′-OH group of a preexisting DNA chain serving as a primer. With most DNA polymerases, DNA is used as the template to guide each incorporation event. However, telomerase and other reverse transcriptases use or RNA as the template. Correct polymerization results in the synthesis of a DNA chain that is complementary to the template strand of DNA.

Figure 2

X-ray crystallographic structures of DNA polymerases reveal common structural motifs representing the palm, fingers, and thumb subdomains that play important roles in nucleotide binding and phosphoryl transfer. The left panel displays the structure of a high-fidelity DNA polymerase (bacteriophage RB69) in the “open” conformation while the right panel displays the structure of the polymerase in the “closed” conformation.

Figure 3

Kinetic mechanism for DNA polymerases. Individual steps along the pathway for DNA polymerization are numbered and identified as described in the text. E, polymerase; DNA_n, DNA substrate; E′, conformational change in DNA polymerase; PP_i, inorganic pyrophosphate; DNA_n+1, DNA product (DNA extended by one nucleobase).

Figure 4

“Trojan Horse” strategy of using nucleoside analogs to inhibit DNA polymerization. The polymerase is provided with a modified nucleotide in which the 3′-OH group required for DNA elongation is missing, replaced with a halogen, or altered in configuration from a normal ribose sugar. Since the nucleobase component is left unmodified, the polymerase incorporates the nucleotide analog into DNA as efficiently as its natural counterpart. After incorporation, the nucleotide lacking a usable 3′-OH group is refractory to elongation causing the induction of apoptosis by the termination of DNA synthesis.

Figure 5

Structures of FDA approved nucleoside analogs. Purine-like nucleosides include cladribine (2-chlorodeoxyadenosine), clofarabine [2-chloro-9-(2′deoxy-2′-fluoroarabinofuranosyl)adenine], fludarabine (9-β-D-arabinoside-2-fluoroadenine), and pentostatin (2′-deoxycoformycin). Pyrimidine-like nucleosides include cytarabine [1-β-D-arabinofuranosylcytosine (Ara-C)], gemcitabine [2′,2′-difluorodeoxycytidine (dFdC)], 5-aza-deoxycytidine, and tezacitabine.

Figure 6

Differences in the mechanism of chain termination by gemcitabine and ara-C. After incorporation into DNA, ara-CTP terminates DNA synthesis directly at the site of incorporation while gemcitabine can be elongated by one additional nucleotide. The placement of gemcitabine at the penultimate position is termed “masked chain termination” since the terminal nucleotide masks detection and removal of gemcitabine by exonucleases or DNA repair enzymes. (A) Normal DNA synthesis. (B) Inhibition by fludarabine. (C) Inhibition by gemcitabine.

Figure 7

Models for the efficient bypass of DNA lesions during translesion DNA synthesis. After encountering a DNA lesion, a replicative DNA polymerase incorporates a nucleotide opposite it but is unable to extend beyond the lesion. A specialized DNA polymerase extends beyond the lesion. Once the lesion is bypassed, the specialized polymerase is replaced by the replicative polymerase to resume processive DNA synthesis. In some cases, the replicative polymerase stalls at the DNA lesion and is unable to incorporate a nucleotide opposite the adduct. As a result, a specialized polymerase is recruited to the DNA lesion to incorporate a nucleotide opposite the lesion. A different specialized polymerase is then recruited for extension beyond the DNA lesion.