Interactions between Quinolones and Bacillus anthracis Gyrase and the Basis of Drug Resistance.

Gyrase appears to be the primary cellular target for quinolone antibacterials in multiple pathogenic bacteria, including Bacillus anthracis, the causative agent of anthrax. Given the significance of this type II topoisomerase as a drug target, it is critical to understand how quinolones interact with gyrase and how specific mutations lead to resistance. However, these important issues have yet to be addressed for a canonical gyrase. Therefore, we utilized a mechanistic approach to characterize interactions of quinolones with wild-type B. anthracis gyrase and enzymes containing the most common quinolone resistance mutations. Results indicate that clinically relevant quinolones interact with the enzyme through a water-metal ion bridge in which a noncatalytic divalent metal ion is chelated by the C3/C4 keto acid of the drug. In contrast to other bacterial type II topoisomerases that have been examined, the bridge is anchored to gyrase primarily through a single residue (Ser85). Substitution of groups at the quinolone C7 and C8 positions generated drugs that were less dependent on the water-metal ion bridge and overcame resistance. Thus, by analyzing the interactions of drugs with type II topoisomerases from individual bacteria, it may be possible to identify specific quinolone derivatives that can overcome target-mediated resistance in important pathogenic species.

Catalytic core of human topoisomerase IIα: insights into enzyme-DNA interactions and drug mechanism.

Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during the catalytic cycle. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To further address the roles of these domains, we analyzed the activity of the catalytic core of human topoisomerase IIα. The catalytic core and the wild-type enzyme both maintained higher levels of cleavage with negatively (as compared to positively) supercoiled plasmid, indicating that the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core alone displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (i.e., substrates that did not contain crossovers). Finally, in contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed us to further characterize the mechanism of etoposide quinone, a drug metabolite that functions primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfacial poison. These results further define the distinct contributions of the N-terminal gate and the catalytic core to topoisomerase II function. The catalytic core senses the handedness of DNA supercoils during cleavage, while the N-terminal gate is critical for capturing the transport segment and for the activity of covalent poisons.

Stimulation of topoisomerase II-mediated DNA cleavage by benzene metabolites.

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic and quinone-based metabolites, especially 1,4-benzoquinone. Since the DNA damage induced by 1,4-benzoquinone is consistent with that of topoisomerase II-targeted drugs, it has been proposed that the compound initiates specific types of leukemia by acting as a topoisomerase II poison. This hypothesis, however, was not supported by initial in vitro studies. While 1,4-benzoquinone inhibited topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage were not observed. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of benzene metabolites (including 1,4-benzoquinone, 1,4-hydroquinone, catechol, 1,2,4-benzenetriol, 2,2'-biphenol, and 4,4'-biphenol) on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. Other metabolites displayed considerably less activity. DNA cleavage enhancement by 1,4-benzoquinone was unseen in previous studies due to the presence of reducing agents and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. Unlike anticancer drugs such as etoposide that interact with topoisomerase IIalpha in a noncovalent manner, the actions of 1,4-benzoquinone appear to involve a covalent attachment to the enzyme. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. These findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of some benzene-induced leukemias.

Topoisomerase II and leukemia.

Type II topoisomerases are essential enzymes that modulate DNA under- and overwinding, knotting, and tangling. Beyond their critical physiological functions, these enzymes are the targets for some of the most widely prescribed anticancer drugs (topoisomerase II poisons) in clinical use. Topoisomerase II poisons kill cells by increasing levels of covalent enzyme-cleaved DNA complexes that are normal reaction intermediates. Drugs such as etoposide, doxorubicin, and mitoxantrone are frontline therapies for a variety of solid tumors and hematological malignancies. Unfortunately, their use also is associated with the development of specific leukemias. Regimens that include etoposide or doxorubicin are linked to the occurrence of acute myeloid leukemias that feature rearrangements at chromosomal band 11q23. Similar rearrangements are seen in infant leukemias and are associated with gestational diets that are high in naturally occurring topoisomerase II-active compounds. Finally, regimens that include mitoxantrone and epirubicin are linked to acute promyelocytic leukemias that feature t(15;17) rearrangements. The first part of this article will focus on type II topoisomerases and describe the mechanism of enzyme and drug action. The second part will discuss how topoisomerase II poisons trigger chromosomal breaks that lead to leukemia and potential approaches for dissociating the actions of drugs from their leukemogenic potential.

Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison.

Although benzene induces leukemias in humans, the compound is not believed to generate chromosomal damage directly. Rather, benzene is thought to act through a series of phenolic- and quinone-based metabolites, especially 1,4-benzoquinone. A recent study found that 1,4-benzoquinone is a potent topoisomerase II poison in vitro and in cultured human cells [Lindsey et al. (2004) Biochemistry 43, 7363-7374]. Because benzene is metabolized to multiple compounds in addition to 1,4-benzoquinone, we determined the effects of several phenolic metabolites, including catechol, 1,2,4-benzenetriol, 1,4-hydroquinone, 2,2'-biphenol, and 4,4'-biphenol, on the DNA cleavage activity of human topoisomerase II alpha. Only 1,4-hydroquinone generated substantial levels of topoisomerase II-mediated DNA scission. DNA cleavage with this compound approached levels observed with 1,4-benzoquinone (approximately 5- vs 8-fold) but required a considerably higher concentration (approximately 250 vs 25 microM). 1,4-Hydroquinone is a precursor to 1,4-benzoquinone in the body and can be activated to the quinone by redox cycling. It is not known whether the effects of 1,4-hydroquinone on human topoisomerase II alpha reflect a lower reactivity of the hydroquinone or a low level of activation to the quinone. The high concentration of 1,4-hydroquinone required to increase enzyme-mediated DNA cleavage is consistent with either explanation. 1,4-Hydroquinone displayed attributes against topoisomerase II alpha, including DNA cleavage specificity, that were similar to those of 1,4-benzoquinone. However, 1,4-hydroquinone consistently inhibited DNA ligation to a greater extent than 1,4-benzoquinone. This latter result implies that the hydroquinone may display (at least in part) independent activity against topoisomerase II alpha. The present findings are consistent with the hypothesis that topoisomerase II alpha plays a role in the initiation of specific types of leukemia that are induced by benzene and its metabolites.

N-acetyl-p-benzoquinone imine, the toxic metabolite of acetaminophen, is a topoisomerase II poison.

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.

1,4-Benzoquinone is a topoisomerase II poison.

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.