Novel Indenoisoquinolines NSC 725776 and NSC 724998 Produce Persistent Topoisomerase I Cleavage Complexes and Overcome Multidrug Resistance

Smitha Antony,Kazutaka Takagi,Andrew Morrell,Mark Cushman,Mollie H Wright,Ana I Robles,Yves Pommier,Muthukaman Nagarajan,Keli K Agama,Lyuba Varticovski,Ze-Hong Miao

doi:10.1158/0008-5472.can-07-0938

Abstract

Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those drug-induced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G(2)-M and a dependence on functional p53 for their cytotoxicity. Dose-dependent gamma-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These gamma-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0).

Highlights

Camptothecin (CPT) derivatives, which selectively target DNA topoisomerase I (Top1; refs. 1–3), are among the most effective anticancer drugs recently approved by the U.S Food and Drug Administration (FDA), especially against solid tumors [4]
To overcome some of the clinical limitations imposed by CPT derivatives, we aim to generate derivatives that (a) are chemically stable, (b) have a distinct DNA cleavage pattern, (c) would produce Top1-DNA cleavage complexes that persist longer in cells than those trapped by CPT, and (d) would be limited substrates for the drug efflux membrane pumps multidrug resistance (MDR)-1 (P-glycoprotein) and ABCG2 compared with the clinical derivatives of CPT [8, 17]
As known Top1 inhibitors, such as CPT or the indenoisoquinoline MJ-III-65 (NSC 706744), induce S-phase cell cycle arrest [4, 13, 38], we evaluated the effect of NSC 725776 and NSC 724998 on cell cycle progression

Summary

Introduction

Camptothecin (CPT) derivatives, which selectively target DNA topoisomerase I (Top; refs. 1–3), are among the most effective anticancer drugs recently approved by the U.S Food and Drug Administration (FDA), especially against solid tumors [4]. They are chemically unstable, as the a-hydroxylactone in the E-ring is rapidly converted to a carboxylate whose tight binding to human serum albumin limits the available active drug [4,5,6]. CPT diffuses rapidly from the Top cleavage. The fact that CPTs are the only class of Top poisons used in the clinic to date prompted us to search for novel Top inhibitors. Following a COMPARE analysis of the National Cancer Institute (NCI) drug screen database using CPT as a seed, we identified an indenoisoquinoline (NSC 314622) as a Top inhibitor NSC 314622 served as a lead compound for the development of indenoisoquinolines to overcome some of the limitations of CPTs and to develop novel classes of Top inhibitors with different anticancer activity profiles. We have obtained crystal structures of two different indenoisoquinolines within Top cleavage complexes [14,15,16]

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