7.05.1 Introduction The Topoisomerase Enzymes

The mammalian topoisomerase enzymes topo I, topo IIa, and topo IIP are regulatory homodimeric enzymes that catalyze the breakage and religation of DNA. They provide swivel points for relaxing the supercoils generated during the transcription and replication of DNA, and by the segregation of chromatids prior to mitosis.1 The topo II enzyme has major isozymes coded by two separate genes.2 The IIa isozyme (170 kDa) maps3 to chromosome 17, is regulated during the cell cycle, and is the target of virtually all of the DNA intercalating topo inhibitors. The IIP form (180 kDa) maps4 to chromosome 3, and becomes the predominant isozyme in both noncycling cells and in cells resistant to 'classical' topo II agents. The gene for the topo I enzyme gene is located on chromosome 20, with the gene copy number varying from two to eight in a panel of colorectal cancer cell lines.5

There are three main steps in the action of these enzymes. The first is binding to recognition sequences on the DNA. These binding sites for topo I and II involve 15-19 and 30 contiguous nucleotide pairs respectively,6'7 although initial binding is probably to a smaller segment of about five base pairs, followed by conformational changes to allow binding to the full site. Enzyme-promoted hydrolysis of a phosphodiester bond then occurs, with the protein becoming covalently bound via a tyrosine hydroxyl group to the 5'-end of the broken DNA chain.8 Topo II generally effects cuts on each strand about five base pairs apart, whereas topo I generates a single-strand cut. The resulting complex, termed the cleavable complex, is held together by the interactions between the enzyme domains, while a second strand (in the case of topo II) is passed through the gap to relieve torsional stress.8 The process is regulated so that minimum time is spent in this fragile state before the breaks are religated in an ATP-driven process.9 Mechanism of Topoisomerase Inhibition by Drugs

A large number of drugs, generally referred to as topo inhibitors, interact with topo enzymes and affect one of these actions. Collectively they are a major class of anticancer agents, and one of the mainstays of current cancer chemotherapy. At high concentrations they can bind to the DNA or the protein, and compete with the formation of the initial ternary enzyme-DNA-drug complex, although this is unlikely to be the primary mechanism of clinical activity for the majority of the drugs. More important is their subsequent interference with the DNA cleavage, DNA strand passing (in the case of topo IIa and IIP), or DNA re-ligation steps. DNA intercalating agents in particular can unwind the double helix and distort DNA structure at the enzyme cleavage sites. This is thought to hinder registration between the cleaved ends of the DNA during re-ligation, leading to accumulation of the cleavable complex and an increase in strand breaks. Such drugs essentially convert the topo enzyme into a DNA damaging agent, and are also termed topo poisons.1 The selective killing of tumor cells by topo inhibitors arises because many tumor cells overexpress these enzymes to enhance cellular proliferation, and the degree of poisoning is a function of the amount of the enzyme present. Classification of Topoisomerase Inhibitors

The primary classification of topoisomerase inhibitors is a functional one; inhibitors of topo I, topo II, or dual topo I/II inhibitors.10 While it would be preferable to fully classify these compounds by their detailed mechanism of action there is in general not enough known to allow this, and the next level of classification is by their mode of DNA interaction. The largest class of drugs by far (and the majority of the topo II and dual topo I/II inhibitors) are the DNA intercalators. Intercalation as a mode of reversible binding of ligands to DNA was first described by Lerman,11 and involves insertion of the ligand between the base pairs. This is now understood to be the major DNA binding mode of virtually any flat polyaromatic ligand of sufficiently large surface area and suitable steric properties, and is driven primarily by stacking (charge-transfer and dipole-induced dipole) and electrostatic interactions, with entropy (dislodgement of ordered water around the DNA) of lesser and variable importance.12 A great deal of work has been done delineating the ligand structural properties that favor intercalation, the geometry, kinetics, and DNA sequence-selectivity of the binding process, and the effect of such binding on the structure of the DNA substrate.13 A much smaller class of compounds (no clinical agents) bind to DNA in the minor groove, and there is a substantial and diverse class of non-DNA binders (compounds with little direct DNA affinity but which nevertheless form ternary complexes). Finally, a number of unrelated compounds affect topoisomerase function by virtue of their effects on the multiple signaling pathways that interact with the topoisomerases, but these compounds are not primarily known as topo inhibitors and will not be covered here. This review notes the important clinical inhibitors of the topo I and/or topo II enzymes, as well as some less clinically successful examples that have been of scientific importance. It focuses on the more recent emerging drugs, which demonstrate how the major drawbacks of these classes of compounds are being addressed.

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