Targeting DNA topoisomerase II in cancer chemotherapy

Abstract

Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.

Figure 1. Mechanisms of inhibiting of Top2

Top2 can be inhibited at several different points in the enzyme reaction cycle, which can generate different biochemical and cellular consequences. One simple mode of inhibition is to inhibit a step early in the enzyme reaction cycle. For example, competitive inhibitors of ATP binding prevent strand passage, and do not generate enzyme mediated DNA damage. While agents such as novobiocin and coumermycin (not shown on the figure) inhibit both prokaryotic and eukaryotic Top2s, they are either less potent as well as relatively nonspecific (e.g., novobiocin) or are poorly taken up by mammalian cells (e.g., coumermycin). Similar effects would occur with inhibitors that prevent the binding of Top2 to DNA such as aclarubicin. Agents that prevent DNA cleavage by Top2, such as merbarone would also be expected to act as simple catalytic inhibitors. While merbarone clearly prevents DNA cleavage by Top2, merbarone clearly affects other targets besides Top2. A second mode of inhibition is blocking the catalytic cycle after DNA is cleaved but prior to DNA religation. This mode of inhibition occurs for most currently used Top2 targeting agents including anthracyclines and epipodophyllotoxins, as well as for agents that target prokaryotic type II topoisomerases. These agents prevent enzyme turnover, and therefore greatly inhibit the enzyme catalytic activity, however, the clearest effect is the generation of high levels of Top2:DNA covalent complexes. Therefore, these inhibitors generate DNA damage, and interfere with many DNA metabolic events such as transcription and replication. Since agents of this class convert Top2 into an agent that induces cellular damage, they have been termed topoisomerase poisons. Top2 can be inhibited after strand passage is completed, but prior to ATP hydrolysis and dissociation of N-terminal dimerization. Bisdioxopiperazines such as dexrazoxane (ICRF-187) inhibit both ATP hydrolysis and maintain Top2 as a closed clamp . As is the case with Top2 poisons, bisdioxopiperazines inhibit Top2 catalytic activity mainly by blocking enzyme turnover. Although these agents are frequently termed catalytic inhibitors, they leave Top2 trapped on DNA, and may interfere with DNA metabolism in ways distinct from the inhibitors described in pathway (A). Nonetheless, since bisdioxopiperazines are relatively specific for Top2, they are the most commonly used catalytic inhibitors of Top2 in mammalian cells .

Figure 2. Structure of Top2 bound to DNA

Dong and Berger have described a structure of the breakage reunion domain of yeast Top2 bound to DNA. As described in the text, a key feature of the structure is the large bend induced in the DNA. Another key feature is the proximity of the TOPRIM domain and the active site tyrosine. Panel A shows the overall structure of yeast Top2 bound to DNA; the DNA is shown in yellow, while the winged helix domain helices are shown in purple. Previous studies have shown that drug resistant mutants occur near both the TOPRIM domain and the active site tyrosine. Residues labeled in blue are amino acids that are altered in drug hypersensitive top2 mutants. Pro473, is distant from the tyrosine in the primary sequence, but is close to Tyr782 in this structrure. Pro473Leu is hypersensitive to the intercalator mAMSA. Gly737 and Ser740 are both in the winged helix domain; Ser740Trp is hypersensitive to etoposide, while G737 is hypersensitive to mAMSA. Panel B shows just the region around the active site Tyr, note the presence of a Mg ion complexed within the TOPRIM domain. The figure is adapted from Rogojina and Nitiss.

Figure 3. Pathways for the repair of Top2 mediated DNA damage

Following recognition of Top2 covalent complexes (perhaps by collision with replication forks), collision with other tracking proteins, such as RNA polymerase, or other undiscovered surveillance processes, repair can be initiated by proteolysis or by nucleolytic processing. Proteolysis will not completely remove the protein, since the phosphotyrosyl linkage to DNA cannot be removed by proteases. Therefore, after proteolysis, a nucleolytic processing step is still required. As illustrated, the product of nucleolytic processing is a DNA molecule containing a double-strand break. Note that Top2 can be trapped as a single strand break, since the two subunits break DNA in an independent, but coordinated process. For simplicity, the trapped structure that is illustrated shows a double strand break. Processing of a covalent complex with a single strand break might generate either a single strand break, or a double strand break. Recent experiments have demonstrated that a Top2 enzyme that can only generate single strand breaks can confer cytotoxicity in the presence of Top2 poisons. In the case of a double strand break, repair is carried out mainly by homologous recombination or non-homologous end-joining. Repair can also take place by error-prone single strand annealing pathways. The error prone repair of Top2 generated DNA double strand breaks can generate translocations that lead to secondary malignancies. Repair of single strand breaks arising from Top2 covalent complexes have not been carefully explored.