Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity

doi:10.1016/S0006-2952(02)00916-4

Biochemical Pharmacology

Volume 63, Issue 9, 1 May 2002, Pages 1653-1662

https://doi.org/10.1016/S0006-2952(02)00916-4 Get rights and content

Abstract

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug–DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed ‘classical’ pattern observed for intercalation, as proved by the DNA topoisomerase I—unwinding experiments, with relatively weak binding affinity (K_i=1.2×10⁵ M⁻¹), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 10⁵ M⁻¹ and binding site size of 2–3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug–DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.

Introduction

IA (see Table 1 for structures) are a group of antitumor compounds synthesized in our laboratory [1], [2]. IA are cytotoxic in vitro against various human and murine cell lines and exhibit in vivo activity against leukemia P388, melanoma B16, ascites colon 26 adenocarcinoma, colon 38 adenocarcinoma, MAC29 colon carcinoma and HT29 colon in mice [3], [4]. The most promising analog, C-1311 (for review see [5]), has been selected for the clinical development by European Organization for Research and Treatment of Cancer (EORTC).

Despite the advanced stage in clinical development, the knowledge of the mechanism of action of IA is limited. C-1311 was shown to accumulate in the cell nucleus [4], [6], [7], to inhibit cellular nucleic acids synthesis [4], [8], and to trap topoisomerase II cleavage complexes [6]. Cell growth inhibition by IA was accompanied by the induction of G₂ block of the cell cycle [9] followed by apoptosis [7]. This pattern suggested that cellular DNA might be a target for the IA drugs. Given the structural resemblance of IA to other anticancer agents, such as anthracenediones, that are known to bind to DNA, it was possible that DNA binding may play a role also in the case of IA.

Preliminary report of this study [8] and the data published by others [4], [7] suggested indeed that IA drugs are capable of binding to DNA. The nature of these interactions and their relevance to cytotoxic and antitumor properties of IA compounds remains unknown. Since targeting cellular DNA is likely to be significant for the cytotoxic and antitumor properties of IA, it became necessary to evaluate in a quantitative and systematic way various aspects of drug–DNA interactions using a coherent set of analogs displaying divergent biological activities.

In this study, we verified by X-ray crystallography that C-1311 contains planar polycyclic aromatic ring system required for the intercalation into DNA. Intercalative DNA binding has been demonstrated for C-1311 by several approaches. Analysis of several IA analogs of C-1311 showed that the ability to interact with DNA is a common attribute of cytotoxic IA drugs. The differences in DNA binding properties, however, do not fully account for the diverse biological activities of the IA drugs studied.

Section snippets

Chemicals

IA were synthesized and purified as described earlier [1], [2]. Stock solutions (1 mM) were prepared in water and stored at −20°. The RPMI-1640 medium, fetal calf serum, simian virus (SV40) supercoiled DNA and human topoisomerase I were obtained from Life Technologies (Paisley, UK). Methyl-[ $^{3} H$ ]-thymidine, 5-[ $^{3} H$ ]-uridine were from Amersham Ltd. (Piscataway, NJ, USA). Calf thymus DNA (ctDNA) (type II) from Sigma (St. Louis, MO, USA) was used after sonication. The concentrations of DNA solutions

Results

The imidazoacridinone derivatives examined in this study have been selected to include analogs with divergent structures, covering a range of cytotoxic and anticancer activities (Table 1). All these compounds share the same core structure of IA with two varying elements: (1) ring substitutions in the position 5, 7 or 8, and (2) diaminoalkyl side chain of variable length.

Discussion

IA are a group of interesting anticancer agents and the lead compound, C-1311, is accepted for phase I clinical trials. Various observations suggested that DNA binding might be involved in the action of IA [4], [7], [8], [22]. The ability to interact with DNA, in general, and specific binding properties such as: (a) mode of interactions, (b) affinity to DNA, (c) DNA duplex stabilization, and (d) kinetics of association/dissociation can influence biological effects of the drugs. This study

Acknowledgements

We are grateful to Dr. Jan M. Woynarowski (Institute for Drug Development, San Antonio, TX) for helpful discussions and critical reading of this manuscript, and to Dr. Jan Kapuścinski (University of Gdańsk, Poland) for his invaluable methodological suggestions. This work was supported in part by the Polish National Committee for Scientific Research (KBN), Grant no. 4PO5F 00612.

References (33)

J. Kapuscinski et al.
Interactions of antitumor agents ametantrone and mitoxantrone (novatrone) with double-stranded DNA
Biochem. Pharmacol.
(1985)
J.D. McGhee et al.
Theoretical aspects of DNA–protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice
J. Mol. Biol.
(1974)
K. Pawlak et al.
The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA
Chem. Biol. Interact.
(1983)
A. Składanowski et al.
Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumor cells
Biochem. Pharmacol.
(1993)
L.A. Hazlehurst et al.
Correlation of DNA reactivity and cytotoxicity of a new class of anticancer agents: aza-anthracenediones
Cancer Lett.
(1995)
A. Składanowski et al.
Relevance of interstrand DNA cross-linking induced by anthracyclines for their biological activity
Biochem. Pharmacol.
(1994)
Z. Mazerska et al.
Enzymatic activation of a new antitumor drug, 5-diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311, observed after its intercalation into DNA
Biochem. Pharmacol.
(2001)
W.M. Cholody et al.
5-(aminoalkyl)amino]imidazo[4,5,1-de]acridin-6-ones as a novel class of antineoplastic agents. Synthesis and biological activity
J. Med. Chem.
(1990)
W.M. Cholody et al.
Chromophore-modified antineoplastic imidazoacridinones. Synthesis and activity against murine leukemias
J. Med. Chem.
(1992)
H. Kusnierczyk et al.
Experimental antitumor activity and toxicity of the selected triazolo- and imidazoacridinones
Arch. Immunol. Therap. Exp.
(1994)

A.M. Burger et al.

Preclinical evaluation of novel imidazoacridinone derivatives with potent activity against experimental colorectal cancer

Br. J. Cancer

(1996)

Z. Mazerska et al.

C-1311

Drugs Fut.

(1998)

A. Składanowski et al.

Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors

Mol. Pharmacol.

(1996)

A.M. Burger et al.

Cellular uptake, cytotoxicity and DNA binding studies of the novel imidazoacridinone antineoplastic agent C-1311

Br. J. Cancer

(1999)

J. Dzięgielewski et al.

Noncovalent binding of potent imidazoacridinones to DNA

Ann. Oncol.

(1996)

E. Augustin et al.

Imidazoacridinones arrest cell-cycle progression in the G₂ phase of L1210 cells

Cancer Chemother. Pharmacol.

(1996)

Cited by (56)

Electrochemical and in silico approaches for liver metabolic oxidation of antitumor-active triazoloacridinone C-1305
2020, Journal of Pharmaceutical Analysis
Citation Excerpt :
The N-dealkylation and dehydrogenation reactions in the dialkylaminoalkylamino side chain, analogous to those described above, were also predicted by in silico analysis for imidazoacridinone derivative, C-1311 [37]. Such substituent is flexible and, after metabolic activation, it may be able to induce interstrand covalent crosslinks in DNA of tumor cells playing a key role in biological (antitumor) activity of these compounds [24,38,39]. Such mechanism mediating the cytotoxicity was also demonstrated in the case of the clinically used antitumor drugs, mitoxantrone and ametantrone [22].
5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) is a promising antitumor compound developed in our laboratory. A better understanding of its metabolic transformations is still needed to explain the multidirectional mechanism of pharmacological action of triazoloacridinone derivatives at all. Thus, the aim of the current work was to predict oxidative pathways of C-1305 that would reflect its phase I metabolism. The multi-tool analysis of C-1305 metabolism included electrochemical conversion and in silico sites of metabolism predictions in relation to liver microsomal model. In the framework of the first approach, an electrochemical cell was coupled on-line to an electrospray ionization mass spectrometer. The effluent of the electrochemical cell was also injected onto a liquid chromatography column for the separation of different products formed prior to mass spectrometry analysis. In silico studies were performed using MetaSite software. Standard microsomal incubation was employed as a reference procedure. We found that C-1305 underwent electrochemical oxidation primarily on the dialkylaminoalkylamino moiety. An unknown N-dealkylated and hydroxylated C-1305 products have been identified. The electrochemical system was also able to simulate oxygenation reactions. Similar pattern of C-1305 metabolism has been predicted using in silico approach. Both proposed strategies showed high agreement in relation to the generated metabolic products of C-1305. Thus, we conclude that they can be considered as simple alternatives to enzymatic assays, affording time and cost efficiency.
Imidazoacridinone antitumor agent C-1311 as a selective mechanism-based inactivator of human cytochrome P450 1A2 and 3A4 isoenzymes
2016, Pharmacological Reports
Citation Excerpt :
Multiple studies on the molecular mode of C-1311 antitumor action revealed that the metabolic activation of this drug by intracellular enzymes might be a prerequisite step in the biochemical mechanism of its action [10]. Activation under enzymatic oxidative conditions resulted in the intercalation and the following covalent binding of the drug to DNA and other cellular macromolecules [11]. Thus, studies on the molecular mechanism of the enzymatic oxidative activation of C-1311 with different liver drug-metabolizing enzymes were investigated.
5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311), a promising antitumor agent that is also active against autoimmune diseases, was determined to be a selective inhibitor of the cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1311 might modulate the effectiveness of other drugs used in multidrug therapy. The present work aimed to identify the mechanism of the observed C-1311-mediated inactivation of CYP1A2 and CYP3A4.
The inactivation experiments were performed in vitro using the human recombinant CYP1A2 and CYP3A4 (Bactosomes). CYP isoenzyme activities were determined using the CYP-specific reactions, 7-ethoxycoumarin O-deethylation (CYP1A2) and testosterone 6β-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by RP-HPLC with UV-Vis detection.
The inhibition of CYPs by C-1311 was time-, concentration- and NADPH-dependent, which suggested a mechanism-based mode of action. Using a 10-fold dilution method and potassium ferricyanide we demonstrated the irreversible nature of the inhibition. In addition, the inhibition was attenuated by the presence of alternate substrates (alternative active site ligands) but not by a nucleophilic trapping agent (glutathione) or a reactive oxygen scavenger (catalase), which further supported a mechanism-based action. Substrate depletion partition ratios of 299 and 985 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.
Our results indicated that C-1311 is a potent mechanism-based inactivator of CYP1A2 and CYP3A4. This finding provided new insights into the mechanism of C-1311 antitumor action, particularly in relation to potential pharmacokinetic drug–drug interactions between C-1311 and/or its derivatives and the substrates of CYP isoforms.
Alteration in DNA binding pattern of conformationally locked NC(O)N system: A spectroscopic investigation
2016, International Journal of Biological Macromolecules
The binding mode of a conformationally locked NC(O)N planar system with deoxyribonucleic acid (DNA) is investigated using various spectroscopic and enzymatic assays. Compound 1 and its four different salts (comp. 2–5) were prepared for this purpose. They showed certain changes in their respective DNA-compound complex at ground state and excited state as measured by UV–vis and fluorescence emission spectra. The Stern-Volmer quenching constant (K_SV) for the neutral species (1) is found 8545 M⁻¹, whereas, for its salts 2, 3, 4 and 5 the quenching constants were 33510 M⁻¹, 11352 M⁻¹, 19693 M⁻¹ and 27270 M⁻¹ respectively. Nevertheless, the binding constant values remain comparable in neutral and salt forms except for 5. To elucidate the reason we took their CD spectra and ran a topoisomerase I (Topo I) assay. These experimental data revel the fact that compound 1 (neutral form) binds at the minor groove of DNA, whereas, its salt (2) has an extended intercalating property.
Importance of retention data from affinity and reverse-phase high-performance liquid chromatography on antitumor activity prediction of imidazoacridinones using QSAR strategy
2012, Journal of Pharmaceutical and Biomedical Analysis
Quantitative structure–activity relationships (QSAR) studies for prediction of cytotoxic and antitumor activity of imidazoacridinones (IA) based on experimentally obtained high-performance liquid chromatography (HPLC) retention data and calculated parameters using computational (molecular modeling) medicinal chemistry methods were proposed. The RP-HPLC and affinity-HPLC chromatographic techniques with four diversified HPLC systems applying columns with octadecylsilanes (C18), phosphatidylcholine (IAM), as well as α₁-glycoprotein (AGP) and albumin (HSA) were used for the determination of the retention constants log k and log k_w which characterize lipophilicity and protein affinity of IA. Moreover, molecular modeling studies were performed using HyperChem and Dragon software's, and structural descriptors were calculated and subsequently used. The QSAR equations using multiple linear regression (MLR) analysis method were derived which indicated that in vivo antileukemia activity of IA depends on cytotoxic activity against leukemia cells, whereas this cytotoxic activity depends on log k and log k_w parameters obtained on all HPLC systems. Moreover, the QSRR equations were derived and indicated that log k and log k_w parameters depend on calculated non-empirical structural parameters. The predictive power of obtained QSAR and QSRR equations allowed the prediction of cytotoxic and antitumor activity of IA and also their HPLC retention parameters. Finally, the equations can be used for prediction of antileukemia activity of IA without the necessity of carrying out experimental measurements.
Substituent effect on the preferred DNA binding mode and affinity of a homologous series of naphthalene diimides
2011, Bioorganic and Medicinal Chemistry Letters
A combination of isothermal titration calorimetry (ITC), topoisomerase I DNA unwinding assays, and ethidium bromide displacement studies were employed to investigate the binding of a homologous series of naphthalene diimides (NDI) to DNA. Our results suggest that the nature of the substituent plays a significant role in both the preferred binding mode and relative binding affinity of the compounds of this study. Only intercalative-type binding (K = 15 ± 3 × 10⁶ M⁻¹) was observed for the NDI with the smallest substituent (trimethyl-ethylamino), while larger members of the series (diethylmethyl-, dipropylmethyl- and dibutylmethyl-ethylamino substituents) adopted an additional binding mode of higher affinity (K₁ = 31 − 78 × 10⁶ M⁻¹).
Polypharmacology: Principles and methodologies
2022, Polypharmacology: Principles and Methodologies

View all citing articles on Scopus

^☆: Preliminary account of these studies has been presented at 9th EORTC-NCI Symposium on New Drug Development, Amsterdam, The Netherlands, 1996.

¹: Present address: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA.

View full text

Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity☆

Abstract

Introduction

Section snippets

Chemicals

Results

Discussion

Acknowledgements

Biochem. Pharmacol.

J. Mol. Biol.

Chem. Biol. Interact.

Biochem. Pharmacol.

Cancer Lett.

Biochem. Pharmacol.

Biochem. Pharmacol.

5-(aminoalkyl)amino]imidazo[4,5,1-de]acridin-6-ones as a novel class of antineoplastic agents. Synthesis and biological activity

J. Med. Chem.

Chromophore-modified antineoplastic imidazoacridinones. Synthesis and activity against murine leukemias

J. Med. Chem.

Experimental antitumor activity and toxicity of the selected triazolo- and imidazoacridinones

Arch. Immunol. Therap. Exp.

Preclinical evaluation of novel imidazoacridinone derivatives with potent activity against experimental colorectal cancer

Br. J. Cancer

C-1311

Drugs Fut.

Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors

Mol. Pharmacol.

Cellular uptake, cytotoxicity and DNA binding studies of the novel imidazoacridinone antineoplastic agent C-1311

Br. J. Cancer

Noncovalent binding of potent imidazoacridinones to DNA

Ann. Oncol.

Imidazoacridinones arrest cell-cycle progression in the G2 phase of L1210 cells

Cancer Chemother. Pharmacol.

Imidazoacridinones arrest cell-cycle progression in the G₂ phase of L1210 cells