Although etoposide is one of the most widely prescribed drugs used for the treatment of human cancers (1
), the specific ring substituents that mediate its interactions with topoisomerase II have been impossible to define. However, the use of STD [1
H]-NMR has enabled drug-enzyme interactions to be characterized at the proton level. Results indicate that protons on the A–ring, B–ring, and pendent E–ring are in close contact with yeast topoisomerase II and human topoisomerase IIα in the binary enzyme-ligand complex. These findings are summarized in , which highlights hydrogens that interact with topoisomerase II in red and those for which no interactions were observed in green.
FIGURE 7 Summary of etoposide substituents that interact with type II topoisomerases. Protons that interact with the enzyme are shown in red, those that do not are shown in green. Hydroxyl protons that were obscured by the water peak and could not be visualized (more ...)
It has long been known that the glycosidic moiety of etoposide plays important physiological roles (1
). The presence of this group keeps etoposide from interacting with tubulin and substitution of a thiophene for the 8”–methyl alters cellular uptake of the drug. Beyond these physiological functions, it is not clear whether the glycosidic moiety of etoposide plays any direct role in enhancing topoisomerase II-mediated DNA cleavage. To this point, no contacts were observed between topoisomerase II and any glycosidic protons and substitution of the thiophene at the 8” position had no effect on DNA scission. It is notable, however, that substitution of the glycosidic moiety with a flexible and charged amino-alkyl side chain, as in TOP-53, significantly enhanced drug-enzyme binding and drug activity against topoisomerase II. Furthermore, contacts were observed between the enzyme and every observable proton of this side chain in the binary complex. Therefore, while the C–4 glycosidic moiety does not appear to mediate interactions between etoposide and topoisomerase II, other substituents at this position have the capacity to influence drug actions against its enzyme target.
Previous studies indicate that E–ring substituents are important for etoposide function (35
). Indeed, as seen in , removal of the 3’– and 5’–methoxyl groups or the 4’–hydroxyl moiety significantly impairs the ability of etoposide to enhance DNA cleavage mediated by yeast topoisomerase II. As determined by STD [1
H]-NMR, every substituent on the E–ring is intimately associated with the protein in the binary complex. Since the E–ring has free rotation about the 1’–linkage to the C–ring, the H2’ and H6’ protons, as well as the 3’– and 5’–methoxyl groups are chemically equivalent and cannot be distinguished by NMR. A previous study by Long and co-workers found that removal of one of the two methoxyl groups had no obvious effect on levels of DNA scission (37
). Thus, as shown in the shaded region in , we propose that contacts between etoposide and topoisomerase II extend to only a portion of the E–ring.
Although the 3’–methoxyl, 5’–methoxyl, and 4’–hydroxyl moieties of the E–ring all are important for etoposide function (35
), none of them appear to contribute significantly to drug-enzyme binding. In fact, when these groups were replaced with hydrogen atoms, NOE signals were observed by STD [1
H]-NMR for all of the resulting protons. This finding suggests that protein associations with the E–ring may be mediated by stacking interactions rather than any specific group on the ring. In the absence of additional structural information, it is not known whether the 3’–methoxyl, 5’–methoxyl, or 4’–hydroxyl groups contribute to drug function through direct interactions with individual amino acids in topoisomerase II, or by subtly changing the overall geometry of the etoposide-enzyme-DNA complex.
In the absence of topoisomerase II, etoposide exhibits little if any ability to bind to DNA (29
). However, the fact that the drug displays a DNA cleavage site specificity (preferring a cytosine at the base immediately 5’ to the scissile bond (5
) implies that etoposide interacts with DNA in the ternary topoisomerase II-drug-nucleic acid complex. If this is the case, which portions of etoposide contact DNA in the active site of topoisomerase II? Given the strong contacts between the enzyme and the A–, B–, and E–rings we believe that these portions of the drug are unlikely to interact with DNA. Furthermore, substitution of a thiophene for the 8”–methyl in teniposide or an amino alkyl chain in TOP-53 does not alter the DNA cleavage specificity of etoposide, and the amino alkyl substitution at the C4 position interacts strongly with topoisomerase II (5
). Consequently, we also believe that the glycoside moiety of etoposide probably does not contact DNA. Therefore, we propose that if any portions of etoposide interact with DNA in the active site of topoisomerase II, the C– and D–rings are the most likely candidates. Future studies with the ternary enzyme-drug-DNA complex will be required to address this hypothesis directly.
Finally, it should be noted that there is a strong correlation between results obtained from STD [1H]-NMR experiments and DNA cleavage assays. Alterations in etoposide substituents that contact the enzyme in the binary complex diminished the ability of the drug to enhance topoisomerase II-mediated DNA cleavage. Conversely, alterations in drug substituents that increased the number of contacts between the enzyme and etoposide raised levels of DNA scission. Therefore, STD [1H]-NMR results with the binary enzyme-drug complex appear to have predictive value that may contribute to the future development of etoposide derivatives with greater activity against topoisomerase II.