Elsevier

Biochemical Pharmacology

Volume 70, Issue 4, 15 August 2005, Pages 511-519
Biochemical Pharmacology

Mechanism of action of a novel “combi-triazene” engineered to possess a polar functional group on the alkylating moiety: Evidence for enhancement of potency

https://doi.org/10.1016/j.bcp.2005.04.037Get rights and content

Abstract

Previous studies showed that SMA41, a 3-methyltriazene termed “combi-molecules” possessing a dual epidermal growth factor receptor (EGFR)/DNA targeting properties induced potent antiproliferative activity against alkylating-agent-resistant cells expressing EGFR in vitro. However, despite its marked potency, its antitumour activity in vivo was significantly hampered by its poor hydrosolubility and the moderate reactivity of its alkylating moiety. To circumvent this problem, we designed the quinazolinotriazene ZRBA1 to contain a N,N-dimethylaminoethyl group grafted to the 3-position of the triazene chain where it could serve both as a water soluble and a more potent alkylating moiety. ZRBA1 exhibited five-fold stronger EGFR tyrosine kinase (TK) inhibitory activity (IC50 = 37 nM) than SMA41, decomposed into a 6-amino-quinazoline FD105 (IC50 = 200 nM) and preferentially blocked EGF- over platelet-derived growth factor (PDGF)-or serum-induced cell growth. ZRBA1 induced DNA damage, concomitantly blocked EGF-stimulated EGFR phosphorylation by a partially irreversible mechanism in MDA-MB-468 breast cancer cells, and induced partially irreversible antiproliferative activity. It also prevented EGFR-mediated MAP kinase activation and, in contrast to FD105 and SMA41, induced high levels of apoptosis. Furthermore, ZRBA1 showed significantly greater antitumor activity (p < 0.05) than SMA41 in the human MDA-MB-468 breast cancer xenograft model. The results in toto indicate that the appendage of N,N-dimethylaminoethyl to combi-triazenes may be an alternative to the reduced hydrosolubility and also to the lack of potency of monofunctional combi-triazenes against resistant tumours.

Introduction

Overexpression of EGFR and its closely related ErbB-2/HER-2 are observed in many human cancers including breast cancer and are correlated with aggressive progression and poor prognosis [1], [2]. We recently reported a novel tumour-targeting strategy termed the “combi-targeting concept” that sought to combine inhibitors of EGFR TK with DNA damaging agents in order to irreversibly block EGF-dependent tumorigenesis [3], [4], [5], [6], [7]. The combi-targeting concept postulates that a combi-molecule termed I-TZ (see Fig. 1;where I represents the inhibitory moiety and TZ the DNA damaging species), designed to behave not only as an inhibitor of a TK but also to be hydrolyzed to another EGFR TK inhibitor (I) and a DNA damaging species should induce more sustained antitumour activity than the reversible inhibitor alone. The first I-TZ studied in vivo was SMA41, a methylating agent that showed a rather moderate activity in an A431 carcinoma of the vulva xenograft model [8]. This was imputed to its poor water solubility and perhaps to the lack of sensitivity of A431 cells that expresses O6-alkylguanine transferase (AGT), a DNA repair enzyme capable of removing the methyl group from the O6-methylguanine adduct by transferring it onto its own cystein residue [9]. In an attempt to develop a tandem approach to these limitations, we designed the quinazolinotriazene ZRBA1 to contain a polar N,N-dimethylaminoethyl group on the alkylating moiety. This was designed to form N,N-dimethylaminoethylguanine adducts which were expected to be poor substrates for the AGT compared with O6-methylguanine (Fig. 1). ZRBA1 was designed to tautomerize to a non-conjugated form that can generate N,N-dimethylethyldiazonium upon hydrolytic cleavage (see Fig. 1B). Moreover, in contrast to usual solubilizing strategies in the quinazoline series that is often based on grafting morpholinoalkyl group to the 6- or 7-positions of the quinazoline ring [10], the appendage of the N,N-dimethylaminoethyl group to the alkylating moiety presents the advantage of not increasing the steric bulk of the molecule, a debility that could decrease its affinity for the ATP binding site of EGFR. Here, we studied the mechanism of action of this novel combi-molecule and compared its in vivo activity with that of its methylating counterpart SMA41 using an MDA-MB-468 human breast cancer xenograft model.

Section snippets

Drug treatment

FD105, SMA41 and ZRBA1 were synthesized in our laboratory according to published procedures [11], [12], [7]. Temozolomide (TEM) was provided by Schering-Plough (Kenilworth, NJ). In all assays, drug was dissolved in DMSO and subsequently diluted in RPMI-1640 containing 10% fetal bovine serum (FBS) (Wisent Inc. St.-Bruno, Canada) or in DMEM containing 10% FBS immediately before the treatment of cell cultures. In all assays, the concentration of DMSO never exceeded 0.2% (v/v).

Cell culture

The human tumour cell

Solubility and hydrolysis of ZRBA1

Solubility testing demonstrated that ZRBA1 could be dissolved at 0.27 g/l, a threshold concentration higher than that of SMA41 that started precipitating at 0.14 g/l.

The conversion of ZRBA1 to its corresponding aminoquinazoline FD105 in serum containing media was confirmed by spectrofluorometry as FD105 was fluorescent (absorption 270 nm, emission 451 nm). The characterization of the released N,N-dimethylethyldiazonium was reported elsewhere [17]. Based upon fluorescence intensity data collected at

Discussion

The combi-targeting concept postulates that the molecules possessing multiple targets should induce greater antitumour activity in refractory tumours than agents with single mechanism of action. To prove this concept, molecules termed combi-molecules were designed to block EGFR (primary target) and to degrade to a DNA damaging agent (secondary target) plus an additional degradation product that retains EGFR TK inhibitory property (the free inhibitor). The first prototype of these

Acknowledgment

We thank the National Cancer Institute of Canada (NCIC) for financial support (Grant #: 013377).

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