Substituents on etoposide that interact with human topoisomerase IIalpha in the binary enzyme-drug complex: contributions to etoposide binding and activity

Abstract

Etoposide is a widely prescribed anticancer agent that stabilizes topoisomerase II-mediated DNA strand breaks. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. A recent study that focused on yeast topoisomerase II demonstrated that the H15 geminal protons of the etoposide A-ring, the H5 and H8 protons of the B-ring, and the H2', H6', 3'-methoxyl, and 5'-methoxyl protons of the E-ring contact topoisomerase II in the binary enzyme-drug complex [ Wilstermann et al. (2007) Biochemistry 46, 8217-8225 ]. No interactions with the C4 sugar were observed. The present study used DNA cleavage assays, saturation transfer difference [ (1)H] NMR spectroscopy, and enzyme-drug binding studies to further define interactions between etoposide and human topoisomerase IIalpha. Etoposide and three derivatives that lacked the C4 sugar were analyzed. Except for the sugar, 4'-demethyl epipodophyllotoxin is identical to etoposide, epipodophyllotoxin contains a 4'-methoxyl group on the E-ring, and 6,7- O, O-demethylenepipodophyllotoxin replaces the A-ring with a diol. Results suggest that etoposide-topoisomerase IIalpha binding is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring. We propose that the E-ring pocket on the enzyme is confined, because the addition of bulk to this ring adversely affects drug function. The A- and E-rings do not appear to contact DNA in the enzyme-drug-DNA complex. Conversely, the sugar moiety subtly alters DNA interactions. The identification of etoposide substituents that contact topoisomerase IIalpha in the binary complex has predictive value for drug behavior in the enzyme-etoposide-DNA complex.

Figure 1

Structures of etoposide and etoposide derivatives that were employed in the present study.

Figure 2

Effects of etoposide derivatives on DNA cleavage and religation mediated by human topoisomerase IIα. Left: Levels of DNA cleavage were expressed as a fold–enhancement over reactions that were carried out in the absence of drug. Assay mixtures contained 0−200 μM etoposide (open squares), DEPT (closed squares), EPT (open circles), or DDEPT (closed circles). Error bars represent the standard deviation of three independent experiments. Center: The ICE bioassay was used to monitor the level of cleavage complexes in human CEM leukemia cells treated with etoposide derivatives. DNA (10 μg) from cultures treated with no compound (None), 10 μM etoposide, 10 μM DEPT, 50 μM EPT or 50 μM DDEPT for 2 h was blotted onto a nitrocellulose membrane and probed with a polyclonal antibody directed against human topoisomerase IIα. Results are representative of three independent experiments. Right: DNA religation was examined in the absence of compound (none, open triangles) or in the presence of 100 μM etoposide (open squares), DEPT (closed squares) EPT (open circles), or DDEPT (closed circles). Error bars represent the standard deviation of three independent experiments.

Figure 3

Interaction of etoposide (top left), DEPT (top right), EPT (bottom left) or DDEPT (bottom right) with human topoisomerase IIα as determined by STD [¹H]-NMR spectroscopy. Difference and off resonance (reference) spectra are shown. Spectra are representative of at least two independent experiments.

Figure 4

Binding of etoposide and derivatives to human topoisomerase IIα. Reaction mixtures contained 20 μM [³H]etoposide and 0−100 μM non-labeled etoposide (open squares), DEPT (closed squares), EPT (open circles), or DDEPT (closed circles). Levels of [³H]etoposide binding to topoisomerase IIα in the absence of competitor drug were set to 1. Error bars represent the standard deviation of at least three independent experiments.

Figure 5

DNA cleavage site utilization by human topoisomerase IIα in the presence of etoposide derivatives. A singly end-labeled linear 4330 bp fragment of pBR322 was used as the cleavage substrate. An autoradiogram of a polyacrylamide gel is shown. DNA cleavage reactions contained no compound (Topo II), 25 μM etoposide, 25 μM DEPT, 250 μM EPT or 250 μM DDEPT. A DNA control is shown in the far left lane. Bands that were present in etoposide-containing reactions that were weak or absent in reactions that contained DEPT are indicated by asterisks and bands that were present in DEPT-containing reactions that were weak or absent in reactions that contained etoposide are indicated by arrowheads. Data are representative of at least three independent experiments.

Figure 6

Summary of etoposide substituents that interact with human topoisomerase IIα. Protons that interact with the enzyme are shown in large bold print, those that do not are shown in small print. Hydroxyl protons were obscured by the water peak and could not be visualized. The blue region on etoposide, including portions of the A–, B– and E–rings, is proposed to interact with topoisomerase IIα in the binary drug-enzyme complex. E–ring substituents highlighted with yellow boxes are important for drug function and interact with the enzyme, but do not appear to contribute significantly to binding (54). It is proposed that portions of the D–ring and/or sugar moiety of etoposide, which are shaded in gray, may interact with DNA in the drug-stabilized topoisomerase IIα-DNA cleavage complex.