Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug;10(8):1490-9.
doi: 10.1158/1535-7163.MCT-10-1043. Epub 2011 Jun 2.

Molecular and Cellular Pharmacology of the Novel Noncamptothecin Topoisomerase I Inhibitor Genz-644282

Free PMC article

Molecular and Cellular Pharmacology of the Novel Noncamptothecin Topoisomerase I Inhibitor Genz-644282

Dhriti Sooryakumar et al. Mol Cancer Ther. .
Free PMC article


Camptothecin derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure. In addition, they exhibit bone marrow and intestinal toxicity, especially in adults, and are drug efflux substrates. Here, we report a novel Top1 inhibitor, Genz-644282. We show that Genz-644282 and its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with camptothecin. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after compound removal than camptothecin. Concentration-dependent and persistent γH2AX formation was readily observed in cells treated with Genz-644282, and was present in greater than 50% of the cell population following 24 hours compound exposure. The compound shows partial cross-resistance in cell lines resistant to camptothecin. These cell lines include the human prostate DU145RC0.1 and the leukemic CEM/C2 cells. Limited cross-resistance to Genz-644282 was also found in the Top1 knockdown colon cancer (HCT116) and breast cancer (MCF7) cell lines and in human adenocarcinoma cells (KB31/KBV1) that overexpress (P-glycoprotein, ABCB1), a member of the ATP-binding cassette family of cell surface transport proteins known to confer MDR. Together, our results provide the first molecular and cellular characterization of Genz-644282 and its clinically relevant metabolites.


Figure 1
Figure 1. Genz-644282, Genz-649974, Genz-649975, Genz-649978 trap Top 1-cleavage complexes
A) Chemical structures of Genz-644282, Genz-649974, Genz-649975, Genz-649978. B) A 3′-end-labeled 117-bp oligonucleotide was reacted with Top1 in the presence or absence of compound at the indicated concentrations. Arrowheads and numbers indicate the positions of DNA fragments cleaved by Top1 in the oligonucleotide. The bases flanking each cleavage site are indicated in parentheses.
Figure 2
Figure 2. Top 1-cleavage complex induction by Genz-644282
A) Schematic representation of the oligonucleotide substrate containing a single Top1-mediated cleavage site. The positions relative to the Top1 cleavage site [between positions (−3) and (+3)] are indicated. B) Comparisons of Top1-mediated DNA cleavage induced by Genz-644282 and CPT with the oligonucleotide substrate shown in panel A. C) Quantitative analysis of cleavage complexes induced by CPT or Genz-644282 are plotted as a function of compound concentrations.
Figure 3
Figure 3. Induction of persistent Top1 cleavage complexes (Top1cc) and DNA-Protein Crosslinks (DPCs) by Genz-644282 in HCT116 cells
A) Top1cc induced by Genz-644282, CPT, and TPT (1 h) measured by Immunocomplex of Enzyme (ICE) assay. DNA fractions were pooled and serial dilutions were blotted. Top1cc were detected using Top1 C21 monoclonal antibody. B) Top1cc induced by Genz-644282 or its metabolites Genz-649974, Genz-649975, and Genz-649978 (1 μM for 1 h). C) Persistent Genz-644282-induced Top1cc following compound removal. After 1 hour of treatment, cells were cultured in compound-free medium and assayed for Top1cc at the indicated time points. D) DNA-Protein Crosslinks (DPCs) induced by treatment with CPT and Genz-644282. Persistence of DPCs at various time points following drug removal is shown. Cells were pre-labeled with 14C-thymidine and treated with either drug. Fraction of DNA remaining on the filter is plotted against time (hours).
Figure 4
Figure 4. Generation and persistence of γH2AX foci in MCF7 and HCT116 cells in response to Genz-644282 treatments
A and B) Immunofluorescence microscopy of γ-H2AX foci formation in HCT116 and MCF7 cells. Cells were treated with Genz-644282 or CPT for 1 hour. Accumulation of γ-H2AX is shown in red, nuclei are shown in green. C) Comparison of γ-H2AX-positive cells induced by CPT or Genz-644282 as analyzed by flow cytometry (PI: propidium iodide). D) Percentage of γ-H2AX-positive cells induced by continuous treatment with 1 μM Genz-644282 or 1 hour of treatment followed by incubation in compound-free medium for the indicated times. HCT116 cells were treated as shown in the left panel. Cells were fixed and incubated with γ-H2AX antibody and PI. The right panel depicts a quantitative comparison of γ-H2AX-positive cells induced by continuous compound exposure versus a 1-hour treatment followed by incubation in compound-free medium.

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

Publication types

MeSH terms