Figure 1.
Voreloxin is a quinolone derivative.
The chemical structures of voreloxin, ciprofloxacin and doxorubicin are shown. The core naphthyridine, quinolone, and anthracycline components are circled. Similarities between the core of the naphthyridine and quinolone are evident, as is the chemical dissimilarity between these two classes of compounds and the anthracyclines.
Figure 2.
Voreloxin poisons topoisomerase II and induces site-selective DNA DSB.
A, CCRF-CEM cells were untreated (No drug) or treated for 4 h with voreloxin (0.1–20 µM), doxorubicin (1 µM) or etoposide (1 or 10 µM), DNA harvested through a caesium chloride pad, DNA quantities normalized, and topoisomerase II levels analyzed following a slot blot using anti-topoisomerase IIα or β antibodies. The immunoblot is a representative of three independent experiments. Quantitative analysis was performed using the Alpha Innotech digital imaging system and each condition was compared relative to the level of cleavage complex induced by 1 µM etoposide. Error bars represent the standard deviations for three independent experiments. B, pBR322 was incubated in vitro with either purified topoisomerase IIα or β and a dose-titration of voreloxin (0.1–10 µM) or etoposide (5 µM). DNA cleavage was assessed using SDS-PAGE, with untreated reaction mix (0) or DNA alone (DNA) as controls. Densitometry quantification of the indicated band, and the sequence surrounding the cleavage site, are shown in Figure S2.
Figure 3.
Topoisomerase II knockdown has a greater effect on G2 arrest induced by etoposide than by voreloxin or doxorubicin.
A, A549 cells were transfected with siRNA targeting topoisomerase IIα (Topo IIα KD), or with scrambled control siRNA for 48 h, when a sample was taken to confirm the knockdown by Western blot. Blank = no siRNA. At 48 h, cells were treated for 16 h with a dose-titration (0.001–3 µM) of voreloxin and stained with BrdU followed by flow cytometry analysis. The percentages of cells in the G2 phase of the cell cycle, calculated from these data, are represented by the line graphs. Data are representative of 6 independent experiments. B, Cells were transfected and treated as described in (A) with a dose-titration (0.001–3 µM) of voreloxin, doxorubicin or etoposide. The same total transfected cell population was split and seeded for treatment with each drug. The percentages of cells in the G2 phase of the cell cycle are represented by the line graphs. Histograms are shown in Figure S3a, S3b, S3c. Data were consistent in 2 independent experiments.
Figure 4.
Voreloxin and the fused phenyl analog intercalate DNA, while the nonplanar phenyl analog does not.
A, Structures of voreloxin and the two analogs are shown, with the thiazole group of voreloxin boxed and the fused phenyl and phenyl rings highlighted by arrow or box, respectively. B and C, Agarose gels stained with ethidium bromide are shown. Intercalation was evaluated by conversion of negatively supercoiled DNA (-SC) into positively supercoiled DNA (+SC) (4B) or the conversion of relaxed plasmid DNA (Rel) to supercoiled molecules (SC) (4C). Control reactions were carried out in the absence of both drug and enzyme (labeled as DNA) or in the absence of drug but containing enzyme (labeled 0). Topoisomerase I concentration was constant. Reactions containing etoposide (100 µM) and ethidium bromide (10 µM) are included as examples of a nonintercalative and intercalative drug, respectively. Data were consistent in two independent experiments.
Figure 5.
Cytotoxicity correlates with the ability of voreloxin and analogs to intercalate DNA.
A549 cells were treated for 72 h with a dose-titration (0.0001–10 µM) of voreloxin or analog, each treatment point performed in triplicate, N = 2, and the inhibition of proliferation analyzed by MTT assay. Error bars represent standard error of the mean (SEM) for the two independent experiments. The potency of the analogs relative to voreloxin was compared using IC50 values. The compound structures are inset, with the fused phenyl and phenyl rings highlighted by arrow or box, respectively.
Figure 6.
Topoisomerase IIα knockdown reduces the G2 arrest induced by the planar voreloxin analog.
A549 cells were transfected with siRNA targeting topoisomerase IIα (Topo IIα KD), or with scrambled control siRNA, for 48 h. At 48 h, cells were treated for 16 h with a dose-titration (0.037–3 µM) of voreloxin or analog and stained with BrdU followed by flow cytometry analysis. Histograms are shown in Figure S6. The percentages of cells in the G2 phase of the cell cycle were calculated and are represented in the line graphs. No G2 arrest was observed with the nonintercalative analog (Figure S6 and Table S3). Data are representative of three independent experiments.