Elsevier

Biochemical Pharmacology

Volume 61, Issue 6, 15 March 2001, Pages 685-694
Biochemical Pharmacology

Enzymatic activation of a new antitumour drug, 5-diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311, observed after its intercalation into DNA

https://doi.org/10.1016/S0006-2952(01)00527-5Get rights and content

Abstract

The imidazoacridinone derivative, C-1311, is a new antitumour agent that exhibits strong antitumour activity against experimental colorectal cancer and has been selected for entry into clinical trial. The compound has previously been shown to have DNA non-covalent binding properties in vitro and to bind irreversibly to DNA of tumour cells. The latter effect has also been observed in a cell-free system, but only in the presence of activated enzymes. The present studies were aimed at finding out whether and in what way the enzymatic activation of C-1311 and its non-covalent binding to DNA influence or depend on each other. Enzymatic activation was performed with a model system containing horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and was followed by UV-VIS spectroscopy and by HPLC with UV-VIS and electrospray ionisation mass spectrometry detection. DNA non-covalent binding was studied in the cell-free system by means of an unwinding assay and UV-VIS spectroscopy. It was shown that C-1311 was oxidised by the HRP/H2O2 system in a manner dependent on the drug:H2O2 ratio. In the case of ratios of 1:3 and 1:5, the reaction gave highly reactive species that were quickly transformed into the further products p2 and p3 that were unable to intercalate into DNA. In the presence of DNA, C-1311 first intercalated into DNA and the intercalated compound was then oxidised. This oxidation was directed to only one product. Therefore, DNA seems to play the role of a “scavenger” of the reactive oxidation product(s) yielded from the intercalated drug and prevents its further deactivation. We conclude that, under the conditions studied, intercalation of C-1311 into DNA is followed by its HRP-mediated activation, giving rise to the intercalated species that might irreversibly bind to DNA. Since peroxidase-type enzymes are present in the cell nucleus, the proposed sequence of events may also be expected to take place in the cellular environment in vivo.

Introduction

15-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (Fig. 1), is a highly active antitumour compound, developed in our Department [1], [2], which is currently undergoing phase I clinical studies. It exhibits strong cytotoxic properties against the cells of solid tumours [3], [4] and high antitumour activity against transplantable tumours in animals: adenocarcinomas of the colon, MAC 15A, MAC 29, and the human xenograft HT29 [4]. This drug also shows some other attractive pharmacological properties: it does not generate oxygen free radicals and therefore is not expected to display strong cardiotoxicity [2]. C-1311 exhibits only limited mutagenic potential [5]. Cellular transport of this agent occurs rapidly and most of the drug accumulates in the nucleus [4], [6], which enables its fast interaction with DNA.

In an attempt to determine the factors underlying the antitumour properties of this drug, studies concerning several aspects of its biological activity have been carried out. It has been found that C-1311 as well as other imidazoacridinones induce the arrest of cell cycle progression in G2 phase [3], [7]. This arrest seems to be the first biological effect observed in cells treated with these agents. It has been shown that C-1311 induces apoptosis in tumour cells [8]. It is also able to inhibit the catalytic activity of DNA topoisomerase II and stimulate the formation of covalent DNA–topoisomerase complexes. The latter effects are observed in a cell-free system as well as in whole cell [6].

Imidazoacridinones bind non-covalently to calf thymus DNA [9], [10], and this effect seems to be necessary but not sufficient for the biological action of these compounds [9]. C-1311 has been shown to bind irreversibly, presumably covalently, to DNA of tumour cells as well as to DNA in a cell-free system. This irreversible binding in the cell-free system has been observed only when the drug is enzymatically activated [11]. It has also been demonstrated that C-1311 forms interstrand cross-links in DNA of tumour cells, and that this effect is not detected in the case of lysates in which enzymes have been inactivated [12]. The above results allowed us to conclude that the metabolic activation of the imidazoacridinone agent, C-1311, is a prerequisite for its biological activity.

In a previous study, we showed that several imidazoacridinone derivatives underwent enzymatic oxidation in the presence of HRP and that the level of this transformation was related to the antitumour activity of these compounds [13]. The rate as well as the type of products of enzymatic oxidation in the case of highly potent 8-hydroxyimidazoacridinones differed from those observed for less active non-hydroxy derivatives. Therefore, the results obtained confirmed that metabolic activation may represent the crucial step in the biological action of these compounds and indicated that imidazoacridinones underwent specific oxidative activation.

Three earlier observations incited us to undertake the present study: (a) non-covalent binding to DNA was necessary for the biological action of imidazoacridinones; (b) irreversible binding to DNA in the cell-free system occurred only in the presence of the HRP/H2O2 system; and (c) metabolic activation of C-1311 was a prerequisite for biologically important DNA cross-linking. Hence, non-covalent binding to DNA and metabolic activation were found to be the initial steps of imidazoacridinone action, and both these phenomena were necessary for the biological activity of C-1311. Therefore, we wondered what the sequence of events was: did the products of metabolic activation interact with DNA or did the oxidative metabolism of C-1311 take place after DNA non-covalent binding of the drug. Determining this sequence was the aim of the present studies.

Here, the HRP/H2O2 system was chosen as a model of metabolic activation because HRP-mediated oxidation had been shown to be relevant to the biological activity of imidazoacridinones [13]. The enzymatic oxidation of C-1311 was followed by UV-VIS spectroscopy and by HPLC with UV-VIS and ESI–MS detection. The interaction with DNA was studied in the cell-free system by a DNA unwinding assay as well as spectroscopically.

Section snippets

Drugs, enzymes, and chemicals

The imidazoacridinone C-1311 was synthesised in the Department of Pharmaceutical Technology and Biochemistry of the Technical University of Gdańsk [1]. HRP, calf thymus DNA, H2O2, SDS, agarose, Tris–HCl, and methanol (HPLC grade) were purchased from Sigma. SV40 viral DNA, human topoisomerase I, and cell culture chemicals were from GIBCO Life Technologies. Proteinase K was obtained from Serva. All other chemicals used were of analytical grade. Ultrapure water (18 MΩ) was used in all experiments.

Enzymatic oxidation in the presence of the HRP/H2O2 system

The HRP-mediated oxidation of C-1311 was performed in the presence of HRP and 1:1, 1:2, 1:3, and 1:5 drug:H2O2 ratios. The samples were subjected to HPLC analysis and the chromatograms obtained are shown in Fig. 2. For equimolar ratio of the substrates, one main product, p1, and small amounts of p2 and p3 were observed. The time-course of the reaction (data not shown) revealed that the concentration of C-1311 dropped twice during the first 10 min and then reached the plateau state, which was

Discussion

The present study was aimed at determining the relationship between the DNA non-covalent binding of imidazoacridinone drug and its capacity for metabolic activation in the model enzymatic system. It is known from previous studies that (a) DNA non-covalent binding of C-1311 is necessary for its biological activity [9] and (b) enzymatic oxidation is necessary for irreversible binding of this drug to DNA in a cell-free system [11]. The summary scheme of the results obtained in this paper is

Acknowledgements

This work was supported by Grants 3 T09A 06713 and 3 T09A17919 from the Committee for Scientific Research (KBN), Poland. This work was presented in part at the European Workshop on Drug Metabolism, Copenhagen, June 1998 and at the European Workshop on Drug Metabolism, St. Andrews, June 2000. We are grateful to Dr. Agnieszka Bartoszek for careful reading of the manuscript.

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