Resveratrol: A novel type of topoisomerase II inhibitor

Abstract

Resveratrol, a polyphenol found in various plant sources, has gained attention as a possible agent responsible for the purported health benefits of certain foods, such as red wine. Despite annual multi-million dollar market sales as a nutriceutical, there is little consensus about the physiological roles of resveratrol. One suggested molecular target of resveratrol is eukaryotic topoisomerase II (topo II), an enzyme essential for chromosome segregation and DNA supercoiling homeostasis. Interestingly, resveratrol is chemically similar to ICRF-187, a clinically approved chemotherapeutic that stabilizes an ATP-dependent dimerization interface in topo II to block enzyme activity. Based on this similarity, we hypothesized that resveratrol may antagonize topo II by a similar mechanism. Using a variety of biochemical assays, we find that resveratrol indeed acts through the ICRF-187 binding locus, but that it inhibits topo II by preventing ATPase domain dimerization rather than stabilizing it. This work presents the first comprehensive analysis of the biochemical effects of both ICRF-187 and resveratrol on the human isoforms of topo II, and reveals a new mode for the allosteric regulation of topo II through modulation of ATPase status. Natural polyphenols related to resveratrol that have been shown to impact topo II function may operate in a similar manner.

Keywords: DNA topoisomerase; conformational change; enzyme inhibitor; inhibition mechanism; protein drug interaction; resveratrol; site-directed mutagenesis.

Figure 1.

Comparison of ICRF-187 and resveratrol, and location of the HsTop2α^T49I ICRF-187 resistance mutation. A, chemical structures of ICRF-187 and resveratrol. The portions of the compounds that resemble each other are boxed in green. B, the ICRF-187-binding site at the dimerization interface of the ATPase domains is shown from a top-down view of the ScTop2 structure (PDB code 4GFH (47)) The two protomers are shown in gold and cyan, and ICRF-187 in green. Residue Thr²⁷, which is homologous to Thr⁴⁹ in HsTop2α and constitutes a known ICRF resistance locus (43, 57), is shown in red. C, conservation of the ICRF-187/resveratrol-binding site among eukaryotic type II topoisomerases. Conservation scores of residues from a multiple sequence alignment of 106 eukaryotic topo II sequences (70) are mapped onto a surface view of one protomer of the ScTop2 ATPase domain bound to ICRF-187(green) and AMPPNP (yellow) (PDB code 1QZR (43) using CONSURF (71). The other monomer is shown as a cream ribbon.

Figure 2.

Resveratrol, like ICRF-187, inhibits supercoil relaxation by topo II. Representative gels show the relaxation of 6 nm negatively supercoiled plasmid by topo II at varying inhibitor concentrations. Negative controls with no ATP (−ATP) show unrelaxed substrate. Reactions were run to partial or near completion for no-drug controls to afford a maximal dynamic range for monitoring the effects of the compounds. A and B, resveratrol (RSV) (A) and ICRF-187 (B) were titrated in 2-fold concentration increments (0, 25, 50, 100, and 200 μm). Negative control lanes (−ATP) contained the highest concentration of inhibitor tested (200 μm). The remaining fraction of supercoiled (SC) substrate was quantified by normalizing the intensity of the supercoiled substrate band in each lane to that of the negative control. Asterisks indicate significant differences as determined by Student's t test (**, p < 0.01; ***, p < 0.001).

Figure 3.

Resveratrol is not a topo II poison. Representative DNA cleavage assays comparing the effects of resveratrol (RSV) to etoposide (a known topo II poison) and ICRF-187. Negatively supercoiled plasmid DNA (6 nm) was incubated for 20 min with 12.5 nm topo II and 1 mm ATP. Plasmid topoisomers were resolved on an agarose gel containing 0.4 μg/ml of ethidium bromide. Plasmid linearized by a restriction enzyme (BamHI) is run in the first lane as a reference. (The etoposide and resveratrol samples for each enzyme were run on the same gel with one linearized reference lane.) A no-protein (−topo) control lane is shown for the highest concentration of drug tested (200 μm), and serves as a reference for supercoiled plasmid. The highest-migrating band represents nicked or open circle plasmid followed by linear (red squiggle), supercoiled, and relaxed plasmid as depicted to the left of the gel images. All three inhibitors were titrated in 2-fold dilutions (0, 25, 50, 100, and 200 μm).

Figure 4.

Resveratrol and ICRF-187 inhibit eukaryotic topo II ATPase activity. Michaelis-Menten curves are shown for reaction rate (μmol of ATP · min⁻¹ · nmol of topo II⁻¹) versus nucleotide concentration. ATP consumption was monitored in an NADH-coupled ATPase assay. Enzyme concentration in all reactions was 100 nm. Sheared salmon sperm DNA (0.2 mg/ml; ∼3000 bp of DNA per enzyme) was added to each reaction for maximum DNA stimulation of ATPase activity. The concentration of inhibitor for each curve is indicated in the legends above the graphs. Error bars reflect the average of three independent runs.

Figure 5.

Resveratrol prevents ATPase domain dimerization. A, the schematic depicts the placement of the donor (Alexa Fluor 555) and acceptor (Alexa Fluor 647) fluorophores on Cys¹⁸⁰ to allow for optimal detection of ATP-gate dynamics. The graph to the right shows example emission spectra. Normalized intensity values represent the emission intensity measured at each wavelength divided by the total emission intensity measured across the entire spectrum. The ratiometric FRET efficiency (E) was calculated from the maximum donor emission (F_D) and maximum acceptor emission (F_A) as shown in the equation. The change in FRET (ΔFRET) was calculated with respect to the FRET efficiency of the apoenzyme sample (E_apo). B, FRET-based monitoring of ATPase domain closure in the presence of different ligands. Emission spectra were collected from solutions containing 200 nm FRET-labeled topo II and the ligands indicated on the x axis after equilibrating for 30 min at room temperature. Asterisks indicate significant differences from the apoenzyme signal (ΔFRET = 0) as determined by Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). C, time-dependent ATPase closure assay comparing AMPPNP and resveratrol order of addition. Emission spectra were collected from solutions containing 200 nm FRET-labeled enzyme at indicated time points immediately after AMPPNP addition. Error bars indicate the average of three independent runs. D, quantification of AMPPNP and resveratrol order of addition assays. 200 nm FRET-labeled enzyme was preincubated with ligand (1) at room temperature for 15 min before addition of ligand (2). Emission spectra were taken 0 and 30 min after addition of second ligand. Concentrations of ligands used were 0.03 mm AMPPNP and 0.2 mm resveratrol (RSV). Asterisks indicate significant differences as determined by Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 6.

The ATPase activity of HsTop2α^T49I is resistant to ICRF-187 and resveratrol inhibition. Michaelis-Menten curves are shown for reaction rate (μmol ATP · min⁻¹ · nmol topoII⁻¹) versus nucleotide concentration. ATP consumption was monitored by an NADH-coupled ATPase assay. The enzyme concentration in all reactions was 400 nm. Sheared salmon sperm DNA (0.5 mg/ml; ∼2000 bp of DNA per enzyme) was added to each reaction to stimulate ATPase activity. The inhibitor concentration for each curve is indicated to the right of each set of graphs. Error bars reflect the average of three independent runs.

Figure 7.

Supercoil relaxation by HsTop2α^T49I is resistant to ICRF-187 and resveratrol inhibition. A, time course supercoil relaxation assays comparing ICRF-187 inhibition of HsTop2α^WT and HsTop2α^T49I. The relaxation of 6 nm negatively supercoiled plasmid by 12.5 nm HsTop2α at varying concentrations of ICRF-187 was initiated by the addition of 1 mm ATP. ICRF-187 was titrated in 2-fold increments (0, 15, 30, and 60 μm). A negative control lane with no enzyme (−topo) that provides a reference for unrelaxed substrate contained 60 μm ICRF-187. B, time course supercoil relaxation assay comparing resveratrol inhibition of HsTop2α^WT and HsTop2α^T49I. The experiment was conducted as per panel (A) with 10 nm enzyme. Resveratrol was titrated in 2-fold increments (0, 100, 200, and 400 μm). The negative control reaction (−topo) contained 400 μm resveratrol. Inhibition of supercoil relaxation at the 10-min time point was quantified by normalizing the intensity of the supercoiled substrate band at each inhibitor concentration to the maximally depleted supercoiled substrate band in the 0 inhibitor lane. Asterisks indicate significant differences as determined by Student's t test (*, p < 0.05).

Figure 8.

Proposed mechanism for resveratrol-dependent inhibition of topo II. Schematic depicting the different manners by which ICRF-187 and resveratrol inhibit topo II, despite binding to a common locus on the enzyme's ATPase domains. Black arrows denote the uninhibited topo II enzymatic cycle and red blocked lines denote inhibitory steps. The domains of topo II are colored as shows in the bottom left legend. The G-segment of DNA is shown in orange and the T-segment is shown in green.