Neil Osheroff

TOPOISOMERASE POISONS : HARNESSING THE DARK SIDE OF ENZYME MECHANISM

Although the one-dimensional sequence of DNA determines the genetic constitution of an organism, topological relationships within the double helix modulate virtually every physiological function of the genome (1, 2). In the cell, the topology of DNA is orchestrated by enzymes known as topoisomerases (1–7). These ubiquitous enzymes are required for replication, transcription, and recombination and play critical roles in chromosome structure, condensation/decondensation, and segregation (1–9). Over the past decade, interest in topoisomerases has expanded beyond the realm of the basic science laboratory into the clinical arena. These enzymes are now known to be the primary cellular targets for some of the most widely prescribed antibiotics and anticancer drugs used in the treatment of human disease (4, 10–19). This review focuses on the mechanism by which drugs alter the catalytic functions of topoisomerases and convert these essential enzymes into lethal cellular weapons.

Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme.

Topoisomerase II is a ubiquitous enzyme that is essential for the survival of all eukaryotic organisms and plays critical roles in virtually every aspect of DNA metabolism. The enzyme unknots and untangles DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. Beyond its physiological functions, topoisomerase II is the target for some of the most active and widely prescribed anticancer drugs currently utilized for the treatment of human cancers. These drugs act in an insidious fashion and kill cells by increasing levels of covalent topoisomerase II-cleaved DNA complexes that are normally fleeting intermediates in the catalytic cycle of the enzyme. Over the past several years, we have made considerable strides in our understanding of the catalytic mechanism of topoisomerase II and the mechanism of action of drugs targeted to this enzyme. These advances have provided novel insights into the physiological functions of topoisomerase II and have led to the development of more efficacious chemotherapeutic regimens and novel anticancer drugs. Considering the importance of topoisomerase II to the eukaryotic cell and to cancer chemotherapy, it is essential to understand its enzymatic function and pharmacological properties. Therefore, this review will discuss the mechanism of action of eukaryotic topoisomerase II and topoisomerase II-targeted drugs.

Topoisomerase II as a target for anticancer drugs: when enzymes stop being nice.

Topoisomerase II is an essential enzyme that plays a role in virtually every cellular DNA process. This enzyme interconverts different topological forms of DNA by passing one nucleic acid segment through a transient double-stranded break generated in a second segment. By virtue of its double-stranded DNA passage reaction, topoisomerase II is able to regulate DNA over- and underwinding, and can resolve knots and tangles in the genetic material. Beyond the critical physiological functions of the eukaryotic enzyme, topoisomerase II is the target for some of the most successful anticancer drugs used to treat human malignancies. These agents are referred to as topoisomerase II poisons, because they transform the enzyme into a potent cellular toxin. Topoisomerase II poisons act by increasing the concentration of covalent enzyme-cleaved DNA complexes that normally are fleeting intermediates in the catalytic cycle of topoisomerase II. As a result of their action, these drugs generate high levels of enzyme-mediated breaks in the genetic material of treated cells and ultimately trigger cell death pathways. Topoisomerase II is also the target for a second category of drugs referred to as catalytic inhibitors. Compounds in this category prevent topoisomerase II from carrying out its required physiological functions. Drugs from both categories vary widely in their mechanisms of actions. This review focuses on topoisomerase II function and how drugs alter the catalytic cycle of this important enzyme.

Etoposide, Topoisomerase II and Cancer

Etoposide is an important chemotherapeutic agent that is used to treat a wide spectrum of human cancers. It has been in clinical use for more than two decades and remains one of the most highly prescribed anticancer drugs in the world. The primary cytotoxic target for etoposide is topoisomerase II. This ubiquitous enzyme regulates DNA under- and overwinding, and removes knots and tangles from the genome by generating transient double-stranded breaks in the double helix. Etoposide kills cells by stabilizing a covalent enzyme-cleaved DNA complex (known as the cleavage complex) that is a transient intermediate in the catalytic cycle of topoisomerase II. The accumulation of cleavage complexes in treated cells leads to the generation of permanent DNA strand breaks, which trigger recombination/repair pathways, mutagenesis, and chromosomal translocations. If these breaks overwhelm the cell, they can initiate death pathways. Thus, etoposide converts topoisomerase II from an essential enzyme to a potent cellular toxin that fragments the genome. Although the topoisomerase II-DNA cleavage complex is an important target for cancer chemotherapy, there also is evidence that topoisomerase II-mediated DNA strand breaks induced by etoposide and other agents can trigger chromosomal translocations that lead to specific types of leukemia. Given the central role of topoisomerase II in both the cure and initiation of human cancers, it is imperative to further understand the mechanism by which the enzyme cleaves and rejoins the double helix and the process by which etoposide and other anticancer drugs alter topoisomerase II function.

The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing

Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.

Mechanism of Quinolone Action and Resistance

Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone–enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix–helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.

MERBARONE INHIBITS THE CATALYTIC ACTIVITY OF HUMAN TOPOISOMERASE IIALPHA BY BLOCKING DNA CLEAVAGE

Merbarone is a catalytic inhibitor of topoisomerase II that is in clinical trials as an anticancer agent. Despite the potential therapeutic value of this drug, the mechanism by which it blocks topoisomerase II activity has not been delineated. Therefore, to determine the mechanistic basis for the inhibitory action of merbarone, the effects of this drug on individual steps of the catalytic cycle of human topoisomerase IIα were assessed. Concentrations of merbarone that inhibited catalytic activity ≥80% had no effect on either enzyme·DNA binding or ATP hydrolysis. In contrast, the drug was a potent inhibitor of enzyme-mediated DNA scission (in the absence or presence of ATP), and the inhibitory profiles of merbarone for DNA cleavage and relaxation were similar. These data indicate that merbarone acts primarily by blocking topoisomerase II-mediated DNA cleavage. Merbarone inhibited DNA scission in a global (rather than site-specific) fashion but did not appear to intercalate into DNA or bind in the minor groove. Since the drug competed with etoposide (a cleavage-enhancing agent that binds directly to topoisomerase II), it is proposed that merbarone exerts its inhibitory effects through interactions with the enzyme and that the drug shares an interaction domain on topoisomerase II with cleavage-enhancing agents.

Type II topoisomerases as targets for quinolone antibacterials: turning Dr. Jekyll into Mr. Hyde.

Quinolones are a very important family of antibacterial agents that are widely prescribed for the treatment of infections in humans. Although the founding members of this drug class had little clinical impact, successive generations include the most active and broad spectrum oral antibacterials currently in use. In contrast to most other anti-infective drugs, quinolones do not kill bacteria by inhibiting a critical cellular process. Rather, they corrupt the activities of two essential enzymes, DNA gyrase and topoisomerase IV, and induce them to kill cells by generating high levels of double-stranded DNA breaks. A second unique aspect of quinolones is their differential ability to target these two enzymes in different bacteria. Depending upon the bacterial species and quinolone employed, either DNA gyrase or topoisomerase IV serves as the primary cytotoxic target of drug action. While this unusual feature initially stymied development of quinolones with high activity against Gram-positive bacteria, it ultimately opened new vistas for the clinical use of this drug class. In addition to the antibacterial quinolones, specific members of this drug family display high activity against eukaryotic type II topoisomerases, as well as cultured mammalian cells and in vivo tumor models. These antineoplastic quinolones represent a potentially important source of new anticancer agents and provide an opportunity to examine drug mechanism across divergent species. Because of the clinical importance of quinolones, this review will discuss the mechanistic basis for drug efficacy and interactions between these compounds and their topoisomerase targets.

Topoisomerase II·Etoposide Interactions Direct the Formation of Drug-induced Enzyme-DNA Cleavage Complexes

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme·drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide· topoisomerase II (rather than etoposide·DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.