Non-intercalative Triterpenoid Inhibitors of Topoisomerase Ii: a Molecular Docking Study

Theoretical flexible docking studies were carried out on a number of triterpenoids previously shown to be in-hibitors of topoisomerase II in order to assess the nature of binding of these non-intercalative inhibitors to the enzyme. The molecular docking results suggest that most of the triterpenoids preferentially bind to the DNA binding site of topoi-somerase II, while a few also bind to the ATP binding site. These results provide some insight into the mode of activity of these cytotoxic natural products.


INTRODUCTION
Topoisomerases are essential enzymes that catalyze modifications to the tertiary structure of DNA. There are two well-characterized classes of human topoisomerases. Topoisomerase I acts by breaking and religating one DNA strand [1], while topoisomerase II involves double-strand breaking [2]. These enzymes serve to relieve DNA twisting and supercoiling, playing key roles in replication, transcription, and recombinant repair. Topoisomerase II is highly expressed in rapidly proliferating cells [3] and is therefore an attractive target for antitumor drugs.
There are two general classes of topoisomerase II targeting drugs: topoisomerase II poisons and topoisomerase II catalytic inhibitors. Topoisomerase II poisons include etoposide, doxorubicin, and mitoxantrone. These compounds serve to stabilize the enzyme-DNA complex (the "cleavable complex") and prevent the enzyme from religating the cleaved DNA [4]. Both doxorubicin and mitoxantrone are DNA intercalating agents [5] whereas etoposide does not bind DNA but rather apparently binds to the ATP binding site of the N-terminal domain of topoisomerase II [6,7].
ATP is a required cofactor for topoisomerase II [2,8,21]. Topoisomerase II uses the energy released by ATP hydrolysis to induce DNA strand passage. In addition, the binding of ATP causes a conformational change of the enzyme from an open form to a closed clamp form. Therefore, ATP binding and hydrolysis result in opening and closing of the topoisomerase II enzyme. Some topoisomerase II inhibitors (e.g., bisdioxopiperazines and coumarins) act by binding to the ATPase domain of the enzyme [8,9]. Potential binding of triterpenoid topoisomerase II inhibitors was also investigated by docking the compounds into the ATP binding sites of the N-terminal domain of topoisomerase II.

RESULTS AND DISCUSSION
The binding energies of the lowest-energy poses for each of the triterpenoid topoisomerase II inhibitors for the DNA binding site (PDB: 1bjt [22] and 2rgr [23]) and the ATP binding sites (PDB: 1qzr, 1pvg, and 1zxm) are summarized in Table 1 (Fig. 3). Mizushina and co-workers [24] found this to be the preferred binding site for unsaturated fatty acids with yeast topoisomerase II. Not surprisingly, the nature of binding of these lipophilic triterpenoids is largely hydrophobic, and the triterpenoids can dock in various orientations in this binding pocket. There are some trends, however. The lowest-energy pose of lupeol (5) is such that it forms hydrogen bonds between the C(3) hydroxyl group of the ligand and the carboxylate of Asp 687 and the guanidi-nium of Arg 690. Ursolic acid (8) and ganoderic acid X (21) occupy analogous orientations. Both betulinic acid and ursolic acid orient themselves in the binding site to form a salt bridge between the carboxylates of the ligands and the ammonium of Lys 700. Fernane 19 and seco-3,4-friedelin (3) have very similar orientations, but no obvious interactions between the carboxylates and nearby amino acid residues. Fomitellic acid A (6), 3 -corosolic acid (15), and dihydrocelastrol (18), dock into the DNA binding site of topoisomerase II, but preferentially occupy different locations than the other triterpenoid ligands (see Fig. 4). The triterpenoid ligands were docked into the ATP binding sites of both Saccharomyces cerevisiae topoisomerase II (two different structures, PDB: 1qzr and 1pvg [25]) and human topoisomerase II (PDB: 1zxm [26]) (see Fig. 5). Most of the triterpenoid ligands showed lower binding (or no binding) affinity for the ATP binding sites. Four triterpenoids, however, seco-3,4-friedelin (3), demethylzeylasterone (12), celastrol (17), and dihydrocelastrol (18), showed stronger binding for the ATP binding sites than for the DNA binding site.
In the ATPase domain of yeast topoisomerase II, demethylzeylasterone (12) (23), known to be a cytotoxic agent [27], but not yet shown to be a topoisomerase II inhibitor, also docks into the same site with the same orientation (see Fig. 6). Tingenone, therefore, would be expected to be a topoisomerase II inhibitor. Friedelane 3 does not have planar rings and therefore binds to the ATP binding site differently than the quinone-methide triterpenoids 12, 17, 18, and 23. Key hydrogen bonding interactions between seco-3,4-friedelin (3) and the protein are between the C(3) carboxylate of the ligand and the amide of Asn 129 as well as the hydroxyl of Ser 128.   Fig. (2). X-ray crystal structure of Saccharomyces cerevisiae topoisomerase II (PDB: 1bjt) [22] with docked ligand, seco-3,4-friedelin (3) in its lowest-energy pose, occupying the DNA binding site.    The binding of the quinone-methide triterpenoids to the ATPase domain of human topoisomerase II is similar to that observed in the yeast. Demethylzeylasterone (12) interacts with Arg 98 and Ser 149, through the C(23) carboxylate. The C(29) carboxylate interacts with Arg 162, Gln 376, Gly 166, and Tyr 165; and the C(6) ketone hydrogen bonds with Tyr 34. Similarly, seco-3,4-friedelin (3) and seco-3,4-taraxerone (4) bind to the ATP binding site of human topoisomerase II through hydrogen bonding between the C(3) carboxylates of the ligands and Asn 150 and Ser 149. The binding energies of the quinone-methide triterpenoids to the ATP binding sites of topoisomerase II are comparable to those calculated (Molegro) for known ATP binders salvicine [28] (average binding energy = -25.2 kcal/mol) or etoposide [6,7] (average binding energy = -22.4 kcal/mol).
There is no discernable trend between the calculated binding energies in this study and the reported topoisomerase II inhibitory activities. This may be due to the different sources of topoisomerase II used (e.g., yeast, human, parasite), or the fact that enzyme inhibitory concentrations have large differences.  [29].

COMPUTATIONAL METHODS
Molecular structures for the triterpenoids were built using SPARTAN '06 for Windows [30] and geometries optimized using the MMFF 94 force field [31]. Protein-ligand docking studies were carried out based on the crystal structure of the DNA-binding and cleavage core (residues 409-1201) of yeast topoisomerase II (PDB: 1bjt) [22], the crystal structure of the DNA-binding and cleavage domain (residues 419-1177) of human topoisomerase II bound to G-segment DNA (PDB: 2rgr) [23], the crystal structure of the Nterminal ATPase region of yeast topoisomerase II bound to dexrazoxane (PDB: 1qzr) and imino-ATP (PDB: 1pvg) [25], and the crystal structure of the ATPase region of human topoisomerase II bound to imino-ATP (PDB: 1zxm) [26]. All solvent molecules, cofactors, and co-crystallized ligands were removed from the structures. Molecular docking calculations for all compounds were undertaken using Molegro Virtual Docker 2.3 [32,33]. Because it is unknown how and where triterpenoids might bind to topoisomerase II, many different sites were examined in order to probe the entire protein structure for 1bjt. For the 2rgr structure, the DNA was removed from the structure and the triterpenoid ligands were docked in the DNA binding site of the protein. In the case of the ATPase region, only the ATP binding pockets of 1qzr, 1pvg, and 1zxm, were modeled. A sphere of radius 15 Å was centered on the binding site in order to allow each ligand to search. Different orientations of the ligands were searched and ranked based on their energy scores.

SUMMARY
A number of triterpenoid natural products have shown potential antitumor activity by inhibition of topoisomerase II. These enzyme inhibitors are not planar molecules and clearly, then, do not intercalate DNA to form stable cleavable complexes with topoisomerase II. The mode of inhibition as revealed by this study may be either to bind to the enzyme at the DNA binding site, preventing DNA binding, or binding to the ATP binding site, conformationally locking the enzyme and thus preventing DNA binding. The calculated binding energies of the triterpenoid inhibitors, however, do not correlate well with experimental inhibitory concentrations.