HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human

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HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human Topoisomerase II# and # James T Wilson, Cole A. Fief, Klarissa Jackson, Susan L Mercer, and Joseph E. Deweese Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00302 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 25, 2017

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Chemical Research in Toxicology

HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human Topoisomerase IIα α and β James T. Wilson,† Cole A. Fief,† Klarissa D. Jackson,†‡ Susan L. Mercer,†‡ and Joseph E. Deweese†¶* †

Lipscomb University College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences. Nashville, TN 37204-3951, USA ‡ Vanderbilt University School of Medicine, Department of Pharmacology. Nashville, Tennessee 37232-0146, USA ¶ Vanderbilt University School of Medicine, Department of Biochemistry. Nashville, Tennessee 37232-0146, USA

* To whom correspondence should be addressed: Joseph E. Deweese Lipscomb University College of Pharmacy One University Park Drive Nashville, TN 37204-3951 Phone: 615-966-7101 Fax: 615-966-7163 Email: [email protected]

Running title: Inhibition of Topoisomerase IIα and β by Oxidized Cannabidiol

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ABSTRACT

Topoisomerase II is a critical enzyme in replication, transcription, and the regulation of chromatin topology. Several anticancer agents target topoisomerases in order to disrupt cell growth. Cannabidiol is a major non-euphoriant, pharmacologically active component of cannabis. Previously, we examined the cannabidiol derivative HU-331 in order to characterize the mechanism of the compound against topoisomerase IIα. In this current work, we explore whether cannabidiol (CBD) impacts topoisomerase II activity, and we additionally examine the activity of these compounds against topoisomerase IIβ. CBD does not appear to strongly inhibit DNA relaxation and is not a poison of topoisomerase II DNA cleavage. However, oxidation of CBD allows this compound to inhibit DNA relaxation by topoisomerase IIα and β without poisoning DNA cleavage. Additionally, we found that oxidized CBD, similar to HU-331, inhibits ATP hydrolysis and can result in inactivation of topoisomerase IIα and β. We also determined that oxidized CBD and HU331 are both able to stabilize the N-terminal clamp of topoisomerase II. Taken together, we conclude that while CBD does not have significant activity against topoisomerase II, both oxidized CBD and HU-331 are active against both isoforms of topoisomerase II. We hypothesize that oxidized CBD and HU-331 act against the enzyme through interaction with the N-terminal ATPase domain. According to the model we propose, topoisomerase II inactivation may result from a decrease in the ability of the enzyme to bind to DNA when the compound is bound to the N-terminus.

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INTRODUCTION DNA Topoisomerases are targeted therapeutically for the disruption of cancer cell growth.1,

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Because of the critical role of topoisomerases in regulating DNA topology

during transcription and replication, many natural products and synthetic compounds have been explored for potential inhibitory activity against topoisomerases.1-3 Humans encode six topoisomerases including two Type II topoisomerases known as topoisomerase IIα and β (TOP2A and TOP2B).4 Type II topoisomerases are necessary for removing knots and tangles in the DNA. Type II topoisomerases act by generating transient, enzyme-bound, double-strand breaks and then passing another double-helix through the break. Agents that target TOP2 in cancer therapy are generally separated into two broad categories: poisons and catalytic inhibitors.1,

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Etoposide is a well-known TOP2

poison, which acts by blocking the enzyme from ligating DNA.2 This results in an accumulation of topoisomerase II-DNA complexes where the enzyme is unable to ligate and release the cleaved DNA. Catalytic inhibitors, on the other hand, act by disrupting other functions of TOP2 including ATP hydrolysis, DNA binding, and enzyme turnover.2 Cannabidiol (CBD) is a phytocannabinoid found in cannabis.5,

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Unlike ∆9-

tetrahydrocannabinol, CBD is non-euphoriant and displays various effects including antipsychotic and anti-inflammatory properties.5, 6 Additionally, numerous derivatives of CBD have been produced including the cannabinoid quinone, HU-331.6 In a previous study, we followed up on reports of the activity of HU-331 against human TOP2A,7-9 and we established the inhibitory mechanism of this compound.10 We found that HU-331 inhibits ATP hydrolysis without increasing DNA cleavage, which was consistent with

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hypotheses and evidence from previous studies. Additionally, we found evidence that HU-331 likely acts in a covalent manner to inactivate TOP2.10 While it is clear that HU-331 has activity, it has been unclear whether CBD or oxidized products of CBD have any activity against TOP2. Evidence from metabolism studies indicates that oxidization can occur both enzymatically and spontaneously.11, 12 Additionally, there have been no published tests looking at the effect of CBD or HU-331 on TOP2B. Therefore, we set out to examine the activity of CBD, oxidized CBD, and HU-331 against TOP2A and TOP2B. As our results demonstrate, CBD is a weak inhibitor of TOP2A- and TOP2Bmediated DNA relaxation. According to previous studies, CBD oxidizes to the hydroxyquinone form in the presence of oxygen.13 Consistent with this, we observed that some of our CBD stocks were found to be oxidized when analyzed by liquid chromatography-mass spectrometry (LC-MS). Oxidized CBD inhibited DNA relaxation by TOP2A and TOP2B and did not poison DNA cleavage. In addition, oxidized CBD could be reduced by the presence of dithiothreitol (DTT), which blocked activity of the compound. Further, we found that oxidized CBD impacted TOP2 in a manner consistent with HU-331. For example, we found that HU-331 inhibits ATPase and inactivates DNA cleavage by TOP2B, as previously found for TOP2A. Finally, we determined that both HU-331 and the oxidized CBD were able to block the N-terminal clamp. We conclude that HU-331 and oxidized CBD act as catalytic inhibitors of TOP2A and TOP2B. EXPERIMENTAL PROCEDURES Enzymes

and Materials. Wild-type TOP2A and TOP2B were expressed in

Saccharomyces cerevisiae JEL1∆top1 cells and purified as described previously.10 The enzyme was stored at -80ºC as a 1 or 1.5 mg/mL (3-4 µM) stock in 50 mM Tris-HCl, pH 5 ACS Paragon Plus Environment


7.7, 0.1 mM EDTA, 750 mM KCl, 5% glycerol, and 40 µM DTT (carried from the enzyme preparation). Negatively supercoiled pBR322 DNA was prepared using a Plasmid Mega Kit (Qiagen) as described by the manufacturer. Etoposide was obtained from Sigma. CBD and HU-331 were obtained from Cayman Chemical (Ann Arbor, MI) and dried down prior to resuspension in 100% DMSO. Drugs were stored at 4°C as 20 mM stock solutions in 100% DMSO. Where denoted, CBD was oxidized in the presence of equimolar concentrations of potassium hydroxide (KOH) with oxygen bubbling for 24 h. LC-MS/MS methods for characterizing CBD samples are described in Supplemental Information. UV absorbance spectra were measured using a BioTek Cytation3 Multimode Imaging Plate Reader (BioTek, Winooski, VT) at 1 nm increments from 230350 nm. Topoisomerase II-Mediated Relaxation of Plasmid DNA. Reaction mixtures contained ~7 nM wild-type human TOP2A or TOP2B, 5 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in 20 µL of 10 mM Tris-HCl, pH 7.9, 175 mM KCl, 0.1 mM Na2EDTA, 5 mM MgCl2, and 2.5% glycerol. Assays were started by the addition of enzyme, and DNA relaxation mixtures were incubated for 15 min at 37°C. DNA relaxation reactions were carried out in the presence of CBD, HU-331, etoposide, or oxidized CBD at concentrations denoted in the experiments. In some experiments, 200 µM DTT or potassium ferricyanide (KFC) were added to the reactions. Reactions were stopped by the addition of 3 µL of stop solution (77.5 mM Na2EDTA, 0.77% SDS). Samples were mixed with 2 µL of agarose gel loading buffer, heated for 2 min at 45°C, and subjected to gel electrophoresis in 1% agarose gels. The agarose gel was then stained in ethidium bromide for 30 min. DNA bands were visualized by UV light and imaged using a Bio6


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Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). DNA relaxation was monitored by the conversion of supercoiled plasmid DNA to relaxed topoisomers. Topoisomerase II-Mediated Cleavage of Plasmid DNA. Plasmid DNA cleavage reactions were performed using the procedure of Fortune and Osheroff.14 Reaction mixtures contained ~140 nM of human TOP2A or human TOP2B and 5 nM negatively supercoiled pBR322 DNA in 20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5% glycerol. Final reaction mixtures contained ~1 µM DTT, which represents the residual DTT carried along from the enzyme preparation. Unless stated otherwise, assays were started by the addition of enzyme, and DNA cleavage mixtures were incubated for 6 min at 37°C. DNA cleavage reactions were carried out in the absence or presence of CBD, oxidized CBD, etoposide, or HU-331. DNA cleavage complexes were trapped by the addition of 2 µL of 2.5% SDS followed by 2 µL of 250 mM Na2EDTA, pH 8.0. Proteinase K was added (2 µL of a 0.8 mg/mL solution), and reaction mixtures were incubated for 30 min at 37°C to digest TOP2. Samples were mixed with 2 µL of agarose gel loading buffer (60% sucrose in 10 mM Tris-HCl, pH 7.9), heated for 2 min at 45°C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-acetate, pH 8.3, and 2 mM EDTA containing 0.5 µg/mL ethidium bromide. Doublestranded DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to linear molecules. DNA bands were visualized by UV light and quantified using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). Inactivation of DNA cleavage was measured using the protocol above for analyzing DNA cleavage. Prior to the initiation of DNA cleavage, ~83 nM TOP2A or 7



~140 nM TOP2B was incubated with DMSO, CBD, oxidized CBD, or HU-331 (at 100 and 200 µM) for 10 min at 37°C. After 10 min, DNA and 100 µM etoposide were added and the cleavage reaction proceeded for an additional 6 min. Reactions were stopped and processed as described above. Double-strand break cleavage in the presence of CBD compounds was quantified relative to the amount of cleavage induced by etoposide alone, and the values were plotted using GraphPad Prism 6 (La Jolla, CA). Data were analyzed used GraphPad Prism 6 (La Jolla, CA), and statistical analysis was performed using a one-way ANOVA followed by a Tukey’s Post-Test Analysis. Thin-Layer Chromatography-Based ATPase Assay. ATP hydrolysis was monitored using thin-layer chromatography (TLC) on a polyethylenimine (PEI) matrix (Merck KGaA, Darmstadt, Germany) as previously described.10 Briefly, reaction mixtures contained 140 nM of TOP2A or ~280 nM of TOP2B, 3 µg negatively supercoiled pHOT-1 DNA (Topogen, Buena Vista, CO), and 1 mM ATP in 20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5% glycerol. Reactions were incubated at 37oC and 4 µL samples were taken out at increasing time points (0-30 min) and spotted on the TLC plate. Reactions were run in the absence (1% DMSO as a control) or presence of 200 µM CBD, oxidized CBD, or HU-331 (TOP2B only). For the TOP2A reactions, DTT was added to examine whether reducing the compounds would block activity. The plate was then placed in 400 mM ammonium carbonate inside the TLC chamber and resolved. Separation of ADP from ATP was imaged using an Alpha Imager system (Santa Clara, CA) and quantified using Alpha Imager software. Data were calculated as the percent of ATP converted to ADP in each reaction. Data were analyzed used GraphPad Prism 6 (La Jolla, CA), and statistical

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analysis was performed using a one-way ANOVA followed by a Tukey’s Post-Test Analysis. Protein N-terminal Clamp Closing Assay. The stabilization of the N-terminal protein clamp was measured using a modified version of a previously described protocol.15-17 Briefly, ~80 nM human TOP2A or ~110 nM human TOP2B and 7 nM pBR322 were incubated for 10 min at 37°C in a total of 50 µL of clamp closing buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM EDTA, and 8 mM MgCl2) in the absence or presence of 1% DMSO or 200 µM CBD, oxidized CBD, or HU-331. Control reactions including DNA only were also run. After 10 min incubation, 2 mM ATP was added and an additional 10 min incubation was carried out at 37°C. Binding mixtures were then loaded into filter baskets containing glass fiber filters (Millipore) that were pre-equilibrated using clamp closing buffer. Filters were spun at low speed (1 krpm) for 60 s. Reactions were then washed in 275 µL clamp closing buffer (low salt), 300 µL of high salt wash (1 M NaCl), and 300 µL of SDS wash (10 mM TrisHCl, pH 8.0, 1 mM EDTA, and 0.5% SDS) heated to 65°C. Baskets were transferred to new tubes after each wash. Eluates were processed through a GeneJET PCR purification kit (Thermo Fisher Scientific, Waltham, MA). Samples were then resuspended in nucleic acid loading buffer (Bio-Rad) and electrophoresed in a 1% agarose TAE gel containing ethidium bromide. Gels were imaged using BioRad ChemiDoc MP Imaging system (Hercules, CA). Supercoiled DNA bands were quantified for low salt, high salt, and SDS wash eluates for each condition and DNA eluting after the SDS wash was calculated as a percentage of the total from all three washes. Data were analyzed used GraphPad Prism 6 (La Jolla, CA), and statistical analysis was performed using a one-way ANOVA followed by a Tukey’s Post-Test Analysis. 9



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RESULTS AND DISCUSSION Oxidation of CBD. Previously, Kogan et. al synthesized HU-331 by the oxidation of CBD (Figure 1).7-9 While HU-331 is a hydroxyl-quinone via oxidation at the 2’ and 5’ positions, multiple sites on CBD are susceptible to oxidation (Figure 1). Previous reports indicate that CBD is photoreactive and oxidized by air.11,

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Accordingly, we observed

that CBD solutions changed from colorless to yellow over time depending on storage conditions (data not shown). Mass spectrometric analysis of colorless and yellow CBD solutions demonstrated that CBD can oxidize in solution to form products consistent in mass and fragmentation with CBD-hydroxyquinone (Supplemental Figures S1 and S2). To oxidize CBD in a reaction mixture, potassium ferricyanide (KFC) was added, and we also analyzed these products using mass spectrometry (Supplemental Figure S3). While this method has been used previously to oxidize compounds, it is only used in the context of a reaction mixture for immediate oxidation.19,

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To generate a more stable

stock of oxidized CBD for use in a series of assays, we used a KOH oxidation method described by Mechoulam and Hanus.11 Using this method, we generated a mixture containing the hydroxyquinone form of CBD (referred to hereafter as oxidized CBD). Quantification of the oxidized CBD compared to parent indicates that products consistent with the cannabinoid quinone are formed by KOH-oxidation (Supplemental Figure S4). The KOH-oxidized CBD has a characteristic violet color compared to the colorless CBD solution (data not shown), and the absorbance spectra of oxidized CBD appears to be transitional between CBD and HU-331 suggesting incomplete oxidation (Supplemental Figure S5). This is reflective of the LC-MS/MS data, which indicates that CBD is still present in the oxidized solution (Supplemental Figures S4 and S6). 10


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Oxidized CBD strongly inhibits TOP2A and TOP2B-mediated DNA relaxation. TOP2 readily relaxes plasmid DNA in the presence of ATP (see DMSO, lane 2, Figure 2). TOP2 poisons, like etoposide (lane 4), and catalytic inhibitors block the ability of the enzyme to relax negatively supercoiled plasmid DNA. We previously found, consistent with other published data, that HU-331 inhibits TOP2A-mediated DNA relaxation.9, 10 As seen in Figure 2, oxidation of CBD results in inhibition of TOP2-mediated DNA relaxation (lane 6). For this assay, the oxidized CBD resulted from spontaneous oxidation. Additionally, reduction of oxidized CBD by DTT decreased the ability of the compound to inhibit TOP2A (lane 7). In contrast, non-oxidized CBD showed no inhibitory activity (see lanes 9 and 10). The addition of the oxidizing agent KFC to an un-oxidized solution of CBD leads to inhibition of relaxation by TOP2A (lane 11). Since KFC does not appear to inhibit relaxation alone (DMSO+KFC, lane 4), the impact on TOP2-mediated DNA relaxation in the CBD+KFC (lane 11) is consistent with the oxidation of CBD to an active form. As mentioned above, KFC does not provide a stable source of oxidized CBD. Therefore, we oxidized CBD in the presence of KOH (Supplemental Figures S4-S6). All subsequent experiments were conducted using KOH-oxidized CBD. Previous studies were performed only with TOP2A.10 Therefore, relaxation assays were performed in the presence of either TOP2A or TOP2B at increasing concentrations of CBD and KOHoxidized CBD (Figure 3). The oxidation of CBD by KOH increases the inhibitory effect such that inhibition is seen at 10 µM whereas the non-oxidized CBD requires higher levels of compound (100-200 µM) to observe similar levels of inhibition. Similar to our previous data with TOP2A, HU-331 inhibits both TOP2A and TOP2B relaxation, and inhibition is observed at HU-331 concentrations comparable to or lower than oxidized 11



CBD. Again, this data supports the ability of HU-331 and oxidized CBD to inhibit relaxation mediated by TOP2A and TOP2B. Therefore, we set out to determine whether oxidized CBD would act as either a TOP2 poison or catalytic inhibitor. CBD and Oxidized CBD do not poison TOP2A and TOP2B-mediated DNA cleavage. Plasmid DNA cleavage assays were used to assess whether CBD or oxidized CBD poison TOP2-mediated DNA cleavage. As seen in Figure 4, no increase in DNA cleavage for TOP2A or TOP2B is seen with CBD or oxidized CBD, consistent with DNA cleavage results in the presence of HU-331. Together with the relaxation data, this finding indicates that oxidized CBD and HU-331 do not inhibit relaxation via poisoning DNA cleavage. Since oxidized CBD acts similarly to HU-331, we next examined the ability of oxidized CBD to disrupt catalytic activity. Oxidized CBD inhibits TOP2 ATPase. TOP2 utilizes ATP during strand passage to maintain association between the N-terminal portion of the enzyme.21 Hydrolysis of the two ATP molecules bound in the ATPase domains occurs during strand passage and release.2 We hypothesize based upon previous data with TOP2A and HU-331 that oxidized CBD would inhibit ATP hydrolysis by TOP2.10 Therefore, we performed ATPase assays with CBD and oxidized-CBD in the presence of both TOP2A and TOP2B (Figure 5). Consistent with relaxation data, CBD shows minimal ability to inhibit ATP hydrolysis, while oxidized CBD has increased inhibitory activity. We also examined TOP2B ATPase activity in the presence of HU-331 and found that this compound inhibits ATP hydrolysis by TOP2B. Again, it appears that HU-331 has a higher impact on TOP2 activity compared to oxidized CBD. Inhibition of ATP hydrolysis suggests interaction with the N-terminal domain. Therefore, we performed additional experiments to examine the effects of these compounds on TOP2. 12


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Oxidized CBD and HU-331 inactivate TOP2. Some compounds that act on TOP2 bind to the enzyme and disrupt its catalytic function.22 HU-331 inactivates TOP2A when incubated with the enzyme prior to the addition of DNA.10 Therefore, we examined whether HU-331 affects TOP2B and whether oxidized CBD inactivates both TOP2A and TOP2B. Since these compounds do not induce DNA cleavage, we added the TOP2 poison etoposide to increase DNA cleavage levels and provide a mechanism for measuring the loss of enzyme activity. Incubation of either enzyme with DMSO or CBD prior to the addition of DNA and 100 µM etoposide do not appear to disrupt DNA cleavage levels (Figure 6). However, oxidized CBD and HU-331 decreased DNA cleavage levels. Consistent with our previous data with HU-331 and TOP2A, these results indicate that the action of oxidized CBD and HU-331 on TOP2A and TOP2B blocks etoposide from poisoning DNA cleavage. In order to block poisoning by etoposide, the compounds likely are acting on the N-terminal domain of TOP2. To test this, we performed a clamp closing assay to determine whether these compounds could stabilize the N-terminal clamp, which impacts DNA binding. HU-331 and oxidized CBD stabilize the N-terminal clamp of TOP2A. The N-terminal ATPase domain of topoisomerase II serves as a “clamp” that maintains protomer contact during strand passage and keeps the transport segment of DNA from leaving the active site prior to strand passage. A number of compounds including the clinically used dexrazoxane (ICRF-187) and other bis-dioxopiperazines stabilize the N-terminal clamp.2, 16 Additionally, the quinone metabolite of etoposide and 1,4-benzoquinone, both topoisomerase II poisons, also have this effect.23 The result of stabilizing the clamp is that topoisomerase II remains “locked” around DNA and the enzyme is unable to release DNA or to bind additional DNA. In order to test whether HU-331 or CBD 13



impacted the N-terminal clamp, we employed a clamp closing assay.23 In this assay, TOP2 binds to plasmid DNA and to the compound (i.e., HU-331 or CBD). Then, the reaction mixture is incubated in the presence of ATP before being applied to a filter. TOP2 binds the filter, and any DNA bound to the enzyme is retained in the filter. A series of washes are used to disassociate the DNA from the enzyme beginning with a “low” salt solution before proceeding to a “high” salt solution and finally an SDS solution. As a final step, the filter is soaked in proteinase K, which digests TOP2 and releases any remaining bound DNA. The fractions collected from each step are resolved via electrophoresis, and the DNA found in each fraction is quantified and compared to the total for all fractions. As seen in Figure 7, DMSO and CBD alone do not significantly stabilize the Nterminal clamp. However, both oxidized CBD and HU-331 result in salt-stable protein complexes. These results are lower than those seen for etoposide quinone and 1,4benzoquinone, which also stabilize the N-terminal clamp.23 The data for HU-331 are consistent with our previous finding that HU-331 appears to decrease the ability of TOP2 to bind to plasmid DNA when the compound is incubated with the enzyme prior to the addition of DNA.10 These experiments were also performed with TOP2B (Figure 7, lower panel), which resulted in a lower level of stabilized N-terminal clamp compared to TOP2A. This could be due to a lower enzyme activity in TOP2B, or it may relate to differences in the two isoforms. However, the results do appear to support a mechanism of action that involves blocking/stabilizing the N-terminal ATPase domain. Taken together with the inactivation data, it is possible that the cannabinoid quinone is blocking the N-terminal clamp when the compound is present before DNA, which may preclude enzyme:DNA binding. 14


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Conclusions CBD is a major component of cannabis, and cannabis has been attributed with anticancer and other medicinal properties.24 With the increasing legalization of cannabis use in various parts of the world, it is important to examine the toxicology and reactivity profiles of the components. Previous work demonstrated that HU-331 has activity against cancer cells and inhibits TOP2A.7-10,

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However, these studies did not clarify

whether CBD, the precursor to HU-331, has any activity against TOP2. The results above indicate that CBD is minimally active against TOP2A and TOP2B. LC-MS analysis of oxidized CBD solutions indicates the presence of a compound with the same mass (m/z 329) and retention time as the authentic HU-331 standard (see Supplemental Figures S1 and S6). Based upon these observations and upon evidence from the literature, we hypothesize that some of the CBD in the spontaneously-oxidized and KOH-oxidized solutions is in a reactive, quinone form.11, 26 Previous studies indicate that CBD-hydroxyquinone (HU-331) forms protein adducts in liver microsomal preparations and glutathione (GSH) conjugates in GSH trapping experiments.27 Additionally, CBD-hydroxyquinone has also been observed to inhibit liver microsome enzyme activity and to inactivate P450 3A11 activity.18,

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This reactivity is

consistent with our previous findings with HU-331 in the presence of TOP2A.10 Similar to HU-331, oxidized CBD is able to inhibit relaxation without poisoning DNA cleavage, but higher concentrations of oxidized CBD are required to reach a similar level of inhibition by HU-331. DNA relaxation inhibition by oxidized CBD is sensitive to the reducing agent DTT, which was a property also seen with HU-331. Additionally, oxidized CBD is able to inhibit TOP2-mediated ATP hydrolysis and inactivate TOP2 15



when incubated with the enzyme prior to the addition of DNA. Finally, a clamp closing experiment demonstrates that both HU-331 and oxidized CBD can stabilize the Nterminal protein clamp of TOP2, which likely prevents enzyme:DNA binding. The fact that oxidized CBD appears to have less effect than HU-331 likely results from an incomplete oxidation of CBD. Therefore, we conclude that the oxidized CBD solutions likely contain the reactive CBD-hydroxyquinone metabolite. Interestingly, these results lead to a critical toxicology point: environmental agents, natural products, and metabolites may interact with TOP2 in ways that inhibit overall enzyme function. We have observed stabilization of the N-terminal clamp of TOP2 by the drug metabolite etoposide quinone, which is distinct from the mechanism of action of etoposide.23 Previous evidence indicates that 1,4-benzoquinone and the polychlorinated biphenyl, 2-(4-chloro-phenyl)-[1,4]benzoquinone, also stabilize the Nterminal clamp of TOP2.23,

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The common feature among these compounds is the

quinone moiety, which bares similarity to the bis-dioxopiperazine rings of the TOP2 inhibitor ICRF-187. We propose that this evidence supports a model in which HU-331 and oxidized CBD can interact with the N-terminal domain of TOP2 to inhibit ATP hydrolysis and DNA relaxation and to stabilize the N-terminal clamp (Figure 8). The mechanism of action of the compounds does not appear to differ between TOP2A and TOP2B, which is reasonable considering the high degree of similarity between the N-terminal domains. Thus, we propose that KOH-oxidized CBD and HU-331 act as catalytic inhibitors of TOP2. From these results, it is not clear what impact CBD may have physiologically on TOP2. The cannabinoid quinone form of CBD has been implicated as a reactive species 16


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that can be generated by purified murine P450 3A11 and can inactivate cytochrome P450 activity.27 Based upon the available evidence, this metabolite may be a small proportion of the total metabolites, and it is unclear whether this would have any appreciable anticancer or toxic effects.29, 30 Therefore, additional work will be needed to explore whether metabolic oxidation of CBD in vivo produces significant levels of the cannabinoid quinone metabolite to impact enzyme and cellular function.

ACKNOWLEDGEMENTS We thank Dr. Anni Andersen for providing the expression vectors for TOP2A and TOP2B. J.T.W. and C.A.F. were participants in the Pharmaceutical Sciences Summer Research Program of the Lipscomb University College of Pharmacy and Health Sciences. We thank Jessica Murray for reviewing the manuscript.

FUNDING SOURCES: This work was funded by Lipscomb University College of Pharmacy and Health Sciences.

ABBREVIATIONS: CBD: cannabidiol; DTT: dithiothreitol; DSB: double-stranded DNA break; EDTA: ethylenediaminetetraacetic; acid; EMSA: electrophoretic mobility shift assay; liquid chromatography-mass spectrometry: LC-MS; potassium ferricyanide: KFC; potassium hydroxide: KOH; SSB: single-stranded DNA break; TOP2: topoisomerase II.

ASSOCIATED CONTENT 17



Supporting Information Supplemental LC-MS/MS Methods and Supplemental Figures S1-S6 showing analysis of LC-MS data are available in the Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

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Figure Legends: Figure 1: Possible Products of CBD Oxidation. Metabolic and non-metabolic oxidation of CBD is known to result in the formation of several products. However, it is unclear whether biological metabolism of CBD can result in the formation HU-331, which has activity against TOP2. The pathway to HU-331 requires oxidation at the 2’ and 5’ positions. However, oxidation may result in multiple oxidation products (see red arrows). Red arrows indicate other potential sites of CBD oxidation, as described by Jiang, et al.12 Figure 2: Examination of the effects of oxidized and reduced forms of CBD on TOP2A-mediated DNA relaxation. Plasmid DNA relaxation gel images in the presence of TOP2A are shown. Relaxed (Rel) and supercoiled (SC) DNA are denoted at left. DNA only (DNA) is shown in the first lane. The next three lanes (lanes 2-4) are reactions in the presence of TOP2A in the presence or absence of 200 µM dithiothreitol (DTT) or potassium ferricyanide (KFC). A reaction with 200 µM etoposide is also shown in the fifth lane (Etop). Reactions with 200 µM Oxidized CBD (lanes 6-8) or CBD (lanes 9-11) in the presence or absence of DTT and KFC are also shown. Results are representative of three independent experiments. Figure 3: Effects of CBD and oxidized CBD on DNA relaxation by TOP2A and TOP2B. Plasmid DNA relaxation assays were performed in the presence of TOP2A (upper gel images) and TOP2B (lower gel images). Positions of relaxed (Rel) and supercoiled (SC) plasmid DNA are denoted at left. DNA only (DNA) and DNA in the presence of TOP2 and ATP (+TII) are shown. Reactions were also performed in the presence of 50 µM of etoposide (Et) and HU-331 (HU). CBD and oxidized CBD were performed at 10, 25, 50, 100, and 200 µM. As a control, a reaction was performed in the presence of 200 µM potassium hydroxide (KOH). This control as also performed with TOP2B with the same result (data not shown). Gels are representative of three or more independent experiments. Figure 4: Impact of CBD and oxidized CBD on TOP2A and TOP2B. Plasmid DNA cleavage reactions were performed in the presence of TOP2A (upper gel) and TOP2B (lower gel). Supercoiled SC), double-strand break (DSB), and single-strand break (SSB) plasmid DNA bands are denoted at left. DNA only (DNA) and DNA in the presence of TOP2 (+TII) are shown in the first two lanes, respectively. Reactions were performed in the presence of 50 µM etoposide, 50 µM HU-331, and 100 or 200 µM of CBD or oxidized CBD (Ox CBD). Gel images are representative of three or more experiments. Figure 5: Oxidized CBD Inhibits ATP Hydrolysis by TOP2A and TOP2B. TOP2A (upper figure) or TOP2B (lower figure) was incubated for increasing time points (10-30 min) in the presence of DMSO (absence of compound) or CBD at 200 µM. Oxidized CBD, as evidenced from mass spec analysis, was also used. Parallel reactions with TOP2A were treated with dithiothreitol (DTT) to reduce oxidized products. TOP2B was also tested in the presence of HU-331. ATP hydrolysis by TOP2 was measured by resolving ATP and ADP on a TLC plate. Asterisks denote statistically significant (**, p < 0.05; *** p < 0.0001) differences between the means of Ox CBD or HU-331 and DMSO at 30 min. Error bars are the standard deviation of the mean of three or more 23 ACS Paragon Plus Environment


independent experiments. Figure 6: HU-331 and Oxidized CBD Inactivate TOP2. TOP2A (top) or TOP2B (bottom) were incubated with 1% DMSO (black), CBD (blue/diagonal striped), oxidized CBD (Ox CBD, purple/vertical striped), or HU-331 (red) at 100 and 200 µM for 10 min prior to the addition of DNA and 100 µM etoposide (Etop). Results were quantified and cleavage in the presence of DMSO was set to 1, and all other values were calculated relative to DMSO. Asterisks denote statistically significant (**, p < 0.05) differences between the means of Ox CBD or HU-331 and CBD at corresponding concentrations. Error bars represent the standard deviation of three independent experiments. Figure 7: HU-331 and Oxidized CBD Impact the N-terminal Clamp of TOP2. A clamp closing assay was performed with DMSO, CBD, oxidized CBD, and HU-331 at 200 µM. Compounds were incubated with TOP2A (upper panel) or TOP2B (lower panel) and plasmid DNA prior to being washed through a filter. Inset: Gel image shows DNA bands from an agarose gel for the flow through of the low salt wash (L), high salt wash (H), SDS wash (S), and Proteinase K (P) treatment of the filter. Graph depicts quantified DNA from each of the latter three steps compared with the total DNA flow through from each experimental condition. Asterisks (***) denote statistically significant (p < 0.0001) differences between the mean HU-331 (for S and P) and DMSO. Error bars represent the standard deviation of three or more independent experiments. Figure 8: Model of Catalytic Inhibition for HU-331 and Oxidized CBD. HU-331 and Oxidized CBD inactivate human TOP2A and TOP2B when present before DNA. Both compounds also inhibit relaxation and ATPase activity. Additionally, both compounds stabilize the N-terminal clamp of TOP2. TOP2 homodimer is shown with one protomer in gray and the other one colored (yellow: ATPase domain/N-gate; orange: transducer; red: TOPRIM domain; blue: DNA binding/core/C-gate). DNA in the active site is shown in green. Image generated using Pymol from PDB ID 4GFH.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human

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