Lynn J. Guziec

Structure-activity study of the interaction of bioreductive benzoquinone alkylating agents with DNA topoisomerase II

Purpose: Quantitative structure-activity studies were performed on a series of benzoquinone mustard (BM) bifunctional alkylating agents to determine whether DNA topoisomerase II (topo II) inhibition was responsible for cell growth inhibition. Methods: Topo II inhibition was evaluated by decatenation and agarose gel electrophoresis assays. Results: The BM compounds were shown to potently inhibit the decatenation activity of topo II. Though BM compounds promoted the formation of protein-DNA complexes in isolated nuclei and cells, this effect was undiminished when levels of topo II varied. The BM compounds had little activity in a topo II-mediated DNA cleavage assay, suggesting that they do not function as topo II poisons. Rather, BM-induced protein-DNA complex formation was likely due to the bifunctional alkylating reactivity of these compounds. Finally, the growth inhibitory properties of these compounds did not correlate with their ability to inhibit topo II, indicating that these compounds did not exert their cellular activity through inhibition of topo II. Some BM compounds reacted very quickly with glutathione and cysteine, likely initially through an electrophilic Michael addition. In the absence of cysteine, the growth inhibitory effects of BM were increased tenfold, indicating the modulatory effect of cysteine sulfhydryl adducts. EPR studies showed that a semiquinone-free radical was produced by some BM compounds. Conclusions: BM compounds likely exert their action through DNA cross-linking and/or by inducing oxidative stress. Although topo II is not a direct target of these agents, this enzyme may play a role in processing the consequences of direct DNA adduction and/or oxidative DNA damage.

Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIalpha-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIalpha decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIalpha covalent complex indicating that they acted as topoisomerase IIalpha poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.

The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds.

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n=1-5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIalpha-mediated cleavage of DNA and inhibition of DNA topoisomerase IIalpha decatenation activities. The results suggest that the bisanthrapyrazoles with n=2-5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents.

Interactions of sulfur-containing acridine ligands with DNA by ESI-MS

The alkylating proficiency of sulfur-containing mustards may be increased by using an acridine moiety to guide the sulfur mustard to its cellular target. In this study, the interactions of a new series of sulfur-containing acridine ligands, some that also function as alkylating mustards, with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/acridine mixtures. The extent of binding observed for the series of sulfur-containing acridines was similar, presumably because the intercalating acridine moiety was identical. Upon infrared multi-photon dissociation (IRMPD) of the resulting oligonucleotide/sulfur-containing acridine complexes, ejection of the ligand was the dominant pathway for most of the complexes. However, for AS4, an acridine sulfide mustard, and AN1, an acridine nitrogen mustard, strand separation with the ligand remaining on one of the single strands was observed. At higher irradiation times, a variety of sequence ions were observed, some retaining the AS4/AN1 ligand, which was indicative of covalent binding.

A Model for NAD(P)H:Quinoneoxidoreductase 1 (NQO1) Targeted Individualized Cancer Chemotherapy.

NQO1 (NAD(P)H:quinoneoxidoreductase 1) is a reductive enzyme that is an important activator of bioreductive antitumor agents. NQO1 activity varies in individual tumors but is generally higher in tumor cells than in normal cells. NQO1 has been used as a target for tumor specific drug development. We investigated a series of bioreductive benzoquinone mustard analogs as a model for NQO1 targeted individualized cancer chemotherapy. We compared the tumor cell growth inhibitory activity of benzoquinone mustard analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of NQO1. We demonstrated that functional groups of different steric size could be used to produce a series of bioreductive antitumor agents that were activated by different levels of NQO1 in tumor cells. This series of drugs could then be used to target cells with specific levels of NQO1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of NQO1. This approach could be used to develop new bioreductive antitumor agents for NQO1 targeted individualized cancer chemotherapy.

Abstract 3510: The molecular docking-based design and biological evaluation of a novel series of bisintercalating DNA-binding bisanthrapyrazole compounds containing amine linkers

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of bisanthrapyrazole derivatives containing amine linkers were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should lack the cardiotoxicity exhibited by this class of antitumor agent. The concentration dependent increase in DNA melting temperature was used to determine the strength of DNA binding and the bisintercalation potential of the newly synthesized analogs. These compounds were also assessed for their ability to inhibit: 1) the growth of the human erythroleukemic K562 cell line; 2) the decatenation activity of DNA topoisomerase IIα; 3) the relaxation activity of DNA topoisomerase I. Finally, the compounds were evaluated as topoisomerase IIα poisons by measuring the topoisomerase IIα-mediated double strand cleavage of DNA. All of the bisanthrapyrazoles inhibited K562 cell growth in the low to submicromolar concentration range and also strongly inhibited the decatenation activity of topoisomerase IIα. However, none of the bisanthrapyrazole compounds act as topoisomerase IIα poisons. In conclusion, a novel group of bisanthrapyrazole compounds were designed, synthesized, and biologically evaluated as potential anticancer agents. Support: CIHR, a Canada Research Chair in Drug Development to B.B.H., a National Institutes of Health grant CA090787 to J.C.Y. and the Dishman Foundation at Southwestern University. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3510.

Abstract 3509: Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binder and intercalator functionalities, have the potential for increased DNA binding affinity and increased cytotoxicity due to their dual mode of DNA binding. In this study DNA minor groove binder netropsin analogs containing either a mono-N-methylpyrrole carboxamide or two units of N-methylpyrrole carboxamide linked to DNA intercalating anthrapyrazoles were synthesized. Two of the hybrid molecules which possessed bis-methylpyrrole moities and amine head groups displayed submicromolar cytotoxicity towards K562 human leukemia cells. The synthesized hybrids were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIα covalent complex, indicating that they acted as topoisomerase IIα poisons. The results indicate that the hybrid agents have higher affinity for DNA than the parent compounds. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazoles and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents. Support: CIHR, a Canada Research Chair in Drug Development, National Institutes of Health grant CA090787 to J.C.Y., the Dishman Foundation at Southwestern University and the Robert A. Welsh Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3509.