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Binding mechanism of anti-cancer chemotherapeutic drug mitoxantrone to DNA characterized by magnetic tweezers
© The Author(s) 2018
- Received: 19 February 2018
- Accepted: 26 June 2018
- Published: 13 July 2018
Chemotherapeutic agents (anti-cancer drugs) are small cytostatic or cytotoxic molecules that often bind to double-stranded DNA (dsDNA) resulting in modifications of their structural and nanomechanical properties and thus interfering with the cell proliferation process.
We investigated the anthraquinone compound mitoxantrone that is used for treating certain cancer types like leukemia and lymphoma with magnetic tweezers as a single molecule nanosensor. In order to study the association of mitoxantrone with dsDNA, we conducted force-extension and mechanical overwinding experiments with a sensitivity of 10−14 N.
Using this method, we were able to estimate an equilibrium constant of association Ka ≈ 1 × 105 M−1 as well as a binding site size of n ≈ 2.5 base pairs for mitoxantrone. An unwinding angle of mitoxantrone-intercalation of ϑ ≈ 16° was determined.
Moreover, we observed a complex concentration-dependent bimodal binding behavior, where mitoxantrone associates to dsDNA as an intercalator and groove binder simultaneously at low concentrations and as a mere intercalator at high concentrations.
- Magnetic tweezers
- Groove binder
Regarding the high morbidity and mortality rate of cancer diseases in the recent decades, the development of cytostatic and cytotoxic chemotherapeutics is highly promoted. Several types of such anti-tumor agents, e.g. anthracycline, bind to DNA polymers in tumor/cancer cells and consequently result in an inhibition of cell growth (cytostatic/antiproliferative activity) or even necrosis (cytotoxic activity). Their heal efficacy depends strongly on the binding mode and the nanomechanism of the DNA-drug interaction. Therefore, a deep and thorough understanding of these biophysical characteristics of chemotherapeutics in the perspective of molecular recognition contributes significantly to the medical regulation and optimization of pharmaceutics.
Here, F, P, L(c), kBT and d represent the applied force, dsDNA persistence length, dsDNA contour length as functions of the drug concentration c, thermal energy and molecular extension of the dsDNA (end-to-end distance), respectively. Furthermore, we acquired reference “hat curves” via overwinding dsDNA to verify the nick-free structure of probed molecules.
At low MTX concentrations up to 3 µM, we discovered successive shifts of the force-extension curves indicating larger dsDNA contour lengths. Interestingly, at the same time the persistence length decreased from about 50 ± 2 to 42 ± 2 nm. Further increasing the drug concentration, merely an increment of the contour length was detected. At a drug concentration of 15 µM, we found a dsDNA-elongation of 27%. In previous work, we were able to categorize the binding mode of a dsDNA-binding agent by its influence on the nanomechanical properties of the host molecule, i.e. an intercalator elongates the dsDNA virtually without affecting the bending stiffness; in contrast, a groove binder only softens the dsDNA . That leads to the conclusion that MTX-dsDNA association exhibits a concentration-dependent bimodal binding mechanism. Primarily, MTX intercalates and groove-binds to dsDNA simultaneously, i.e. the planar anthraquinone ring interacts with the dsDNA base pairs in both intercalating and groove-like binding modes. Moreover, the aminoethylamino side chains bind electrostatically to the negatively charged phosphate backbones strengthening the MTX-dsDNA interaction. This matches with the results from the earlier reports [14–19, 22, 39–41]. Beyond the threshold concentration of 3 µM, the intercalation becomes dominant. Notably, in the case of bimodal binding, it is still not clear in which groove the electrostatic interaction occurs. Lown et al. and Wang et al. suggested that two aminoethylamino chains fit to the major groove by electrochemical experiments and a high-field 1H-NMR analysis, respectively [14, 18, 20]. In contrast, Mazerski et al. reported a minor-groove association of both side chains . Several other work found that the helically shaped chains of MTX can associate in both grooves. However, the interaction in the minor groove was found less favorable and sequence-selective [15, 16, 19].
Determination of binding mechanism
In addition, we approximated the fractional elongation data to the non-cooperative McGhee-von Hippel binding model (Fig. 2c) and obtained an elongation per intercalated drug molecule of ∆x = 0.37 ± 0.02 nm, corresponding to a rise of a B-DNA base pair (0.34 nm). The binding site size n was determined as n = 2.51 ± 0.11 bp, which is typical for a monointercalator and conforms to the “nearest neighbor exclusion principle” [42–44]. This matches very well with previous results [18, 21, 40] although earlier Kapuscinski et al. also reported a n-value of 5 bp for MTX . Analogously, we calculated an equilibrium constant of association of Ka = (0.98 ± 0.06) × 105 M−1, which is consistent with the results of Kapuscinski et al. of Ka = 2.5 × 105 M−1  but somewhat lower than published by other groups [15, 18, 22–25, 39]. However, since MTX apparently presents a more complex bimodal binding mode, the theoretical model might be of a somewhat limited applicability.
The overwinding experiments were recorded with added MTX concentrations up to 28 µM. The hat curve of bare dsDNA was taken as reference (black curve, Fig. 3a). By increasing the MTX concentration, an obvious shift of the hat curves to negative rotation numbers was observed, indicating a DNA unwinding and further supporting the intercalative binding mode of MTX . In addition, a height increment of the hat curves implies an intercalation induced dsDNA elongation that is fully consistent with our extension experiments .
Moreover, we evaluated and plotted the change in the rotation number ΔR and the elongation of the dsDNA contour length ΔL (Fig. 3b). The linear approximation of the data gave us a slope of 0.121 ± 0.002 turns/nm.
In summary, we investigated the nanomechanical binding mechanism of MTX to dsDNA at room temperature in PBS buffer by employing a MT single molecule nanosensor. As a conventional mono-intercalator, MTX displayed a fast equilibrium assembly compared with bis-intercalators and threading intercalators [53–58]. By means of extending and overwinding individual DNA molecules, we observed an elongation, softening and untwisting of the DNA double helix upon MTX binding in a concentration dependent manner. Based on earlier findings , we identified a bimodal association mode, i.e. MTX exhibits simultaneously an intercalative and groove-binding behavior. In addition, we determined a threshold concentration of 3 µM at which the primary bimodal association declines and mere intercalation becomes dominant. Furthermore, we estimated a binding site size of n ≈ 2.5 bp, which corresponds to the results of previous reports (n = 2.6–3.0 bp) [18, 21, 40]. An elongation of Δx ≈ 0.37 nm induced by each drug molecule was estimated, which is typical for a mono-intercalator, since the bond between the drug molecule and DNA base pairs is stabilized through π-stacking. Moreover, we found that each intercalated MTX molecule unwinds the native DNA helix with an angle θ of about 16°, compensating the elongation-induced tension. Finally, the equilibrium constant of association of MTX-dsDNA interaction was determined to be about Ka ≈ 1 × 105 M−1, which is significantly lower than in previous reports [15, 18, 22–25, 39]. However, other anthraquinone derivates like DRAQ5 were found to occupy a similar binding affinity to DNA [33, 59–63]. The results of this work help to further characterize and quantify the biophysical binding mode of mitoxantrone to dsDNA and in turn support the medical regulation processes.
DA, KT, MR and YW designed the study. KT and YW contributed substantially to the sample preparation. DK performed all measurements and analyzed the data. DK, YW and DA wrote the manuscript. All authors read and approved the final manuscript.
We gratefully acknowledge Helene Schellenberg and Christoph Pelargus for the technical supports.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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This work is financially supported by the Project AN 370/8-1 from the Deutsche Forschungsgemeinschaft as well as by the Strategy Funds of Bielefeld University.
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