Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities

Abstract

We recently reported that the antitumor triazoloacridone, compound C-1305, is a topoisomerase II poison with unusual properties. In this study we characterize the DNA interactions of C-1305 in vitro, in comparison with other topoisomerase II inhibitors. Our results show that C-1305 binds to DNA by intercalation and possesses higher affinity for GC- than AT-DNA as revealed by surface plasmon resonance studies. Chemical probing with DEPC indicated that C-1305 induces structural perturbations in DNA regions with three adjacent guanine residues. Importantly, this effect was highly specific for C-1305 since none of the other 22 DNA interacting drugs tested was able to induce similar structural changes in DNA. Compound C-1305 induced stronger structural changes in guanine triplets at higher pH which suggested that protonation/deprotonation of the drug is important for this drug-specific effect. Molecular modeling analysis predicts that the zwitterionic form of C-1305 intercalates within the guanine triplet, resulting in widening of both DNA grooves and aligning of the triazole ring with the N7 atoms of guanines. Our results show that C-1305 binds to DNA and induces very specific and unusual structural changes in guanine triplets which likely plays an important role in the cytotoxic and antitumor activity of this unique compound.

Figure 1

Chemical structures of triazoloacridones C-1305 and C-1533.

Figure 2

Absorption (A) and CD spectra (B) of C-1305 in the presence of increasing concentrations of DNA (calf thymus DNA, [poly(dA–dT)]₂ and [poly(dG–dC)]₂). DNA titration of the drug was performed in BPE buffer at pH 7.1. To 1 ml of drug solution of 20 µM for absorption measurement and 50 µM for CD spectra, aliquots of a concentrated DNA solution were added. Vertical arrows indicate the increase of phosphate-DNA/drug ratio, horizontal arrows correspond to bathochromic shifts.

Figure 3

Absorption (A) and CD spectra (B) of C-1533 in the presence of increasing concentration of DNA (calf thymus DNA, [poly(dA–dT)]₂ and [poly(dG–dC)]₂). DNA titration of the drug were performed in BPE buffer at pH 7.1. To 1 ml of drug solution at 20 µM for absorption measurement and 50 µM for CD spectra, aliquots of a concentrated DNA solution were added. Vertical arrows indicate the increase of phosphate-DNA/drug ratio.

Figure 4

ELD data for drug binding to DNA. Dependence of the reduced dichroism ΔA/A on the wavelength (A) and electric field strength (B) for DNA (open square), m-AMSA (closed square) and triazoloacridone derivatives C-1305 (open circle) and C-1533 (closed circle). Conditions: 13.6 kV/cm, P/D = 20 (200 µM DNA and 10 µM drug) (A) and ΔA/A was measured in absorption band of 440 nm for the DNA–amsacrine complexes (P/D = 20), 430 nm for the DNA–triazoloacridone derivative complexes (P/D = 20) and 260 nm for the DNA alone (B). All measurements were performed in 1 mM sodium cacodylate buffer, pH 7.0.

Figure 5

SPR sensograms (A) for binding of C-1305 to the [GC]₄ DNA hairpin oligomer in HBS-EP buffer at 25°C. Table (B) represents equilibrium binding constants of C-1305 and C-1533 to [AT]₄ and [GC]₄ DNA.

Figure 6

DEPC reactivity on the 176 bp DNA fragment. Experiments were done in the presence of increasing concentrations of C-1305 and C-1533. Digestion products were resolved on 8% polyacrylamide gel containing 7 M urea. Control tracks contained no drug. Numbers on the left side of the gel refer to the standard numbering scheme for the nucleotide sequence of the DNA fragment based on comparison to the position of the guanines on guanine-specific track. Arrows indicate dose-dependent sequence-specific interaction of C-1305 in guanine-rich regions of DNA. G, guanine track; D, DNA unmodified; C, control DNA.

Figure 7

Effect of DNA topoisomerase inhibitors on DEPC reactivity toward the 176 bp DNA fragment. Digestion products were resolved on 8% polyacrylamide gel containing 7 M urea. Control tracks contained no drug. Numbers on the left side of the gel refer to the standard numbering scheme for the nucleotide sequence of the DNA fragment based on comparison to the position of the guanines on guanine-specific track.

Figure 8

(A) Sequence-dependent stabilization of the DNA secondary structure in the presence of C-1305. The graph represents variation of the melting temperature differences ΔT_m ($T_m^{\text{drug–DNA complex}}-T_m^{\text{DNA alone}}$, in degrees) determined for complexes between C-1305 and different DNA substrates (see Table 2 for oligonucleotide sequences) in BPES buffer pH 7.1. Gray and black bars correspond to drug–DNA ratios of 0.5:1 and 1:1, respectively. (B) Sequence-dependent binding affinity of C-1305 obtained by the competition microdialysis assay in BPES buffer. All experiments were carried out in duplicates at least twice. Results are presented as means ± SD.

Figure 9

Effect of triazolo- and imidazoacridone derivatives on DEPC reactivity toward a 176 bp DNA fragment (A) Optical density analysis of specific drug–DNA interaction sites. (B) Arrow indicates the position of the band on which the cleavage intensity for each compound was measured by densitometry. The graph represents an increase of the cleavage at guanine sites compared to control/untreated DNA.

Figure 10

Influence of pH on DEPC reactivity toward a 176 bp DNA fragment incubated in the presence of C-1305 (A) Experiments were carried out in 10 mM Tris–HCl/NaCl buffer with increasing pH value (ranging from 5.55 to 9.0). Control tracks with DNA (D) were added for each pH value and contained no drug. G, guanine track; C, DNA unmodified; D, control DNA. 1, 20–1 and 20 µM C-1305. Optical density analysis of specific drug–DNA interaction sites. (B) Arrow indicates the position of the band on which the cleavage intensity for each compound was measured by densitometry. The graph represents an increase of the cleavage at guanine site over control/untreated DNA.

Figure 11

Formation of the hydrogen bond between the O4′ atom of the ribose in the ‘upper’ cytidine (arrow) and deprotonated hydroxyl group at position 8 of C-1305 of the zwitterionic form of the drug (a) and protonated terminal nitrogen atom in the side chain of C-1305 (b). Arrow marks O4′ atom of deoxyribose in the ‘upper’ cytosine of the G-triplet. Structures of the DNA fragment without drug and C-1305:DNA complexes (A) where the compound is intercalated inside the G-triplet as a zwitterionic form (B) and with protonated terminal nitrogen of the side chain only (C). Guanine residues from the G-triplet are colored by name, nitrogen atoms N7 of guanines in blue. Compound C-1305 is shown in yellow with nitrogens from triazole ring are marked blue while hydroxyl oxygen is marked red, other nucleotides of the DNA fragment are indicated in cyan. The hydroxyl group at position 8 of the drug chromophore is marked with an arrow. The distribution of MEP on equipotential surfaces for the DNA (A′) and both studied C-1305:DNA complexes (B′ and C′). Colors correspond to potential values, negative potential at −5 kcal/mol is shown in red, positive potential at +5 kcal/mol is shown in blue.