The indenoisoquinoline noncamptothecin topoisomerase I inhibitors: update and perspectives

Abstract

Because camptothecins are effective against previously resistant tumors and are the only class of topoisomerase I (Top1) inhibitors approved for cancer treatment, we developed the indenoisoquinolines. Like camptothecins, the indenoisoquinolines selectively trap Top1-DNA cleavage complexes and have been cocrystallized with the Top1-DNA cleavage complexes. Indenoisoquinolines show antitumor activity in animal models. They have several advantages over the camptothecins: (a) They are synthetic and chemically stable. (b) The Top1 cleavage sites trapped by the indenoisoquinolines have different genomic locations, implying differential targeting of cancer cell genomes. (c) The Top1 cleavage complexes trapped by indenoisoquinolines are more stable, indicative of prolonged drug action. (d) They are seldom or not used as substrates for the multidrug resistance efflux pumps (ABCG2 and MDR-1). Among the >400 indenoisoquinolines synthesized and evaluated, three have been retained as leads for clinical development by the National Cancer Institute: NSC 706744, NSC 725776 (Indimitecan), and NSC 724998 (Indotecan). The trapping of Top1 cleavage complexes by indenoisoquinolines in cells results in the rapid and sustained phosphorylation of histone H2AX (γ-H2AX). We discuss the use of γ-H2AX as a pharmacodynamic biomarker for the clinical development of the indenoisoquinolines.

Figure 1

Chemical structures of camptothecin and selected indenoisoquinolines.

Figure 2

(A) Topoisomerase I (Top1) is an abundant nuclear enzyme. It is mostly associated non-covalently with chromatin. (B) Top1 relaxes DNA by making single-strand breaks that are generated by the covalent linkage of Top1 to the 3'-end of DNA. (C) Camptothecin or indenoisoquinolines bind reversibly to the Top1-DNA cleavage complex and slow down DNA religation. (D) Ternary complex including Top1 (yellow), DNA (dark blue ribbons), and indenoisoquinolines or camptothecins (green and red in the middle). (E) Same structure except Top1 is in ribbon representation. (F) Hydrogen bond network between camptothecin and Top1 amino acid residues. (G). Hydrogen bond network between the indenoisoquinoline derivative MJ-238 and Top1. Note that mutation of asparagine 722 to serine (N722S), which confers resistance to camptothecin and only partially to indenoisoquinolines, is also present in camptothecin-producing plants.

Figure 3

γ-H2AX induction by NSC 725776 and NSC 724998 in human colon cancer HT29 cells. A) Rapid induction of γ-H2AX foci after 1 h treatment with 1 µM of CPT, NSC 725776 or NSC 724998. B) Persistent γ-H2AX signal induced by indenoisoquinolines. After 1 h drug treatments, HT29 cells were further incubated in drug-free medium for 24 h. C) Representative image of a single cell with typical γ-H2AX damage foci. D) Representative image of a single cell with diffuse pan-nuclear staining, which typically develops several hours after drug exposure. Fixed cells were stained with mouse anti-γ-H2AX antibody and goat anti-mouse antibody conjugated with AlexaFluor 488 (green). Nuclei were stained with PI (red).