Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2–3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood, but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ~5–fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.

Fentanyl rose to prominence as an alternative analgesic to morphine nearly 50 years ago; today, fentanyl has re-emerged as a dangerous recreational substance. The increased potency and analgesic effect of fentanyl are advantageous in the treatment of pain but are also responsible for the rise in unintentional opioid overdose deaths. In response to this crisis, fentanyl, its analogues, and even precursors are under heightened regulatory scrutiny. Despite this controversial history, derivatization of fentanyl has resulted in numerous synthetic analogues that provide valuable insights into opioid receptor binding and signaling events. In this review, the impact of fentanyl on chemical neuroscience is shown through its synthesis and properties, manufacturing, metabolism, pharmacology, approved and off-label indications, adverse effects, and the responsibility it has in the opioid epidemic.

Topoisomerases are essential enzymes that are involved in DNA metabolism. Topoisomerase II generates transient DNA strand breaks that are stabilized by anticancer drugs, such as doxorubicin, causing an accumulation of DNA damage. However, doxorubicin causes cardiac toxicity and, like etoposide and other topoisomerase II-targeted agents, can induce DNA damage, resulting in secondary cancers. The cannabinoid quinone HU-331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off-target toxicity than that of doxorubicin. Reports indicate that HU-331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We employed biochemical assays to study the mechanism of action of HU-331 against purified topoisomerase IIα. These assays examined DNA binding, cleavage, ligation, relaxation, and ATPase activities of topoisomerase IIα. Our results demonstrate that HU-331 inhibits topoisomerase IIα-mediated DNA relaxation at micromolar levels. We find that HU-331 does not induce DNA strand breaks in vitro. When added prior to the DNA substrate, HU-331 blocks DNA cleavage and relaxation activities of topoisomerase IIα in a redox-sensitive manner. The action of HU-331 can be blocked, but not reversed, by the presence of dithiothreitol. Our results also show that HU-331 inhibits the ATPase activity of topoisomerase IIα using a noncompetitive mechanism. Preliminary binding studies also indicate that HU-331 decreases the ability of topoisomerase IIα to bind DNA. In summary, HU-331 inhibits relaxation activity without poisoning DNA cleavage. This action is sensitive to reducing agents and appears to involve noncompetitive inhibition of the ATPase activity and possibly inhibition of DNA binding. These studies provide a promising foundation for the exploration of HU-331 as a catalytic inhibitor of topoisomerase IIα.

Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationship development to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2–3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ∼4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ∼2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ∼2–4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIβ.

As the major psychoactive agent in opium and direct precursor for heroin, morphine is a historically critical molecule in chemical neuroscience. A structurally complex phenanthrene alkaloid produced by Papaver somniferum, morphine has fascinated chemists seeking to disentangle pharmacologically beneficial analgesic effects from addiction, tolerance, and dependence liabilities. In this review, we will detail the history of morphine, from the first extraction and isolation by Sertürner in 1804 to the illicit use of morphine and proliferation of opioid use and abuse disorders currently ravaging the United States. Morphine is a molecule of great cultural relevance, as the agent that single-handedly transformed our understanding of pharmacognosy, receptor dynamics, and substance abuse and dependence disorders.

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme/DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to that of etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme/DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-terminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: (1) interfacial poison and (2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA.

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