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 inhibitors 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 topoisomerase 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]. The catalytic inhibitors, on the other hand, block the catalytic activity of DNA topoisomerase II but do not stabilize the DNA-topoisomerase II cleavable complex [5, 8]. Examples of catalytic topoisomerase II inhibitors include the anthracycline aclarubicin, the polyanionic compound surname, the coumarin novobiocin, and bisdioxopiperazines such as sobuzoxane and dexrazoxane [9]. These agents inhibit the catalytic activity of topoisomerase II by preventing the binding of the enzyme to DNA. A number of natural and semisynthetic triterpenoids have shown topoisomerase II inhibitory activity. These include 3,4-seco-8 H-ferna-4(23),9(11)dien-3-oic acid (1) and its corresponding alcohol derivative (2) [10]; seco-3,4-friedelin (3), seco-3,4-taraxerone (4), lupeol (5) [11]; fomitellic acids A and B (6 and 7) [12]; ursolic acid (8), oleanolic acid (9), betulinic acid (10), acetyl boswellic acid (11) [13]; demethylzeylasterone (12) [14]; *Address correspondence to this author at the Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA; Tel: 256824-6519; Fax: 256-824-6349; E-mail: mailto:[email protected] dihydrobetulinic acid (13) [15]; corosolic acid (14), 3 corosolic acid (15), 3 -corosolic acid lactone (16) [16]; celastrol (17), dihydrocelastrol (18) [17]; dehydrotrametononic acid (19), dehydroebriconic acid (20) [18]; ganoderic acid X (21) [19]; and the semisynthetic lanostane derivative (22) [20]. In this study, molecular docking techniques have been used to examine the potential binding sites of these known triterpenoid inhibitors of topoisomerase II in order to probe the possible mechanism of enzyme inhibition. 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. The lowest-energy docking poses for most of the triterpenoids is the DNA binding site of topoisomerase II (see Figs. 1 and 2), including 1-3, 5-14, 16, and 19-22. The key amino acid residues at this binding site are Arg 690, Asp 687. Gln 599, Gln 739, Gln 743, Glu 738, Glu 831, Gly 737, Gly 832, Ile 833, Lys 598, Lys 700, Phe 595, Ser 691, Thr 596, and Trp 597 (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 guanidi14 The Open Bioactive Compounds Journal, 2008, Volume 1 William N. Setzer 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). This alternative binding site is defined by Ala 830, Asn 756, Asn 828, Asp 697, Gln 703, Gln 739, Gln 750, Glu 831, Gly 698, Gly 747, Gly 829, Gly 832, Ile 758, Ile 825, Leu 748, Leu 760, Lys 700, Met 824, and Phe 699. The key interactions involved in docking dihydrocelastrol are a salt bridge between the carboxylate of the ligand and the ammonium moiety of Lys 700, hydrogen bonding between the C(2) hydroxyl group of the ligand and the carbonyl of Gly 829. Interestingly, seco-3,4-taraxerone (4) preferentially docks into an altogether different site (Fig. 4) in the DNA binding region of topoisomerase II, in contrast to the other seco-3,4-triterpenoids, 1-3. This binding site is defined by Ala 722, Ala 725, Ala 742, Ala 777, Ala 778, Ala 779, Ala 780, Arg 781, Gln 743, Glu 589, Glu 590, His 593, Ile 746, Pro 726, Ser 740, and Val 721. The key interaction in the docked pose is a salt bridge between the carboxylate of the ligand and His 593. 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) forms salt bridges between the C(23) carboxylate and the ammonium group of Lys 11 and the guanidinium group of Arg 77, as well as a hydrogen bond with Ser 128; hydrogen bonds between the C(29) carboxylate with the amide hydrogens of Arg 141, Gln 365, Gly H H HO2C H H H H H HO2C H H HO2C H