Tài liệu Báo cáo khoa học: Covalent binding to glutathione of the DNA-alkylating antitumor agent,

Covalent binding to glutathione of the DNA-alkylating antitumor

agent, S23906-1

Marie-He´ le` ne David-Cordonnier1

, William Laine1

, Alexandra Joubert1

, Christelle Tardy1

,

Jean-Franc¸ois Goossens2

, Mostafa Kouach3

, Gilbert Briand3

, Huong Doan Thi Mai4

, Sylvie Michel4

,

Francois Tillequin4

, Michel Koch4

, Ste´ phane Leonce5

, Alain Pierre5 and Christian Bailly1

1

INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Lille, France; 2

Laboratoire de

Chimie Analytique, Faculte´ desSciencesPharmaceutiqueset Biologiques, et 3

Laboratoire de Spectrome´trie de Masse,

Universite´ de Lille, Lille, France; 4

Laboratoire de Pharmacognosie, Universite´ Rene´ Descartes (Paris 5), CNRS UMR8638,

Faculte´ des Sciences Pharmaceutiques et Biologiques, Paris, France; 5

Division Recherche Cance´rologie,

Institut de Recherches SERVIER, Croissy sur Seine, France

The benzoacronycine derivative, S23906-1, was character￾ized recently as a novel potent antitumor agent through

alkylation of the N2 position of guanines in DNA. We show

here that its reactivity towards DNA can be modulated

by glutathione (GSH). The formation of covalent adducts

between GSH and S23906-1 was evidenced by EI-MS, and

the use of different GSH derivatives, amino acids and

dipeptides revealed that the cysteine thiol group is absolutely

required for complex formation because glutathione disul￾fide (GSSG) and other S-blocked derivatives failed to react

covalently with S23906-1. Gel shift assays and fluorescence

measurements indicated that the binding of S23906-1 to

DNA and to GSH are mutually exclusive. Binding of

S23906-1 to an excess of GSH prevents DNA alkylation.

Additional EI-MS measurements performed with the mixed

diester, S28053-1, showed that the acetate leaving group at

the C1 position is the main reactive site in the drug: a reaction

scheme common to GSH and guanines is presented. At the

cellular level, the presence of GSH slightly reduces the

cytotoxic potential of S23906-1 towards KB-3-1 epidermoid

carcinoma cells. The GSH-induced threefold reduction of

the cytotoxicity of S23906-1 is attributed to the reduced

formation of lethal drug–DNA covalent complexes in cells.

Treatment of the cells with buthionine sulfoximine, an

inhibitor of GSH biosynthesis, facilitates the formation of

drug–DNA adducts and promotes the cytotoxic activity.

This study identifies GSH as a reactant for the antitumor

drug, S23906-1, and illustrates a pathway by which GSH

may modulate the cellular sensitivity to this DNA alkylating

agent. The results presented here, using GSH as a biological

nucleophile, fully support our initial hypothesis that DNA

alkylation is the major mechanism of action of the promising

anticancer drug S23906-1.

Keywords: glutathione; DNA alkylation; acronycine; anti￾cancer drug; mechanism of action.

Introduction

The alkaloid acronycine (Fig. 1) was first isolated fromthe

bark of Acronychia baueri (also known as Sarcomeli￾cope simplicifolia), a Rutaceous tree widely distributed in

Australia [1,2]. This tetracyclic alkaloid was shown to be

moderately cytotoxic to a wide range of tumor cells in vitro

[3,4] and to display antitumor activities in vivo [5]. However,

clinical testing of acronycine itself showed a poor response

and the development of this compound was arrested in

the early 1980s. Nevertheless, the antitumor potential of

acronycine has stimulated the synthesis of numerous

analogues [6–8]. Recently, the benzoacronycine derivative,

S23906-1 (Fig. 1), was identified as a potent anticancer drug

with activity against a variety of human tumor xenograft

models in mice [9,10]. S23906-1 has been selected for

advanced preclinical evaluation.

Froma mechanistic point of view, S23906-1 was recently

characterized as a DNA alkylating agent reacting irrevers￾ibly with guanine residues at the N2 position in double￾stranded DNA [11]. The covalent binding to DNA is

apparently responsible for the cytotoxic action [12] and the

capacity of the drug to trigger apoptosis in tumor cells

[13,14]. In the course of our ongoing studies aimed at

characterizing the interaction of S23906-1 with biologically

significant molecules, the reaction with glutathione (GSH)

was examined. The observation that a tricyclic analogue

of S23906-1 (i.e. 1,2-dihydroxy-1,2-dihydroacronycine

diacetate) reacts covalently with benzyl mercaptan to form

Correspondence to C. Bailly, INSERM U-524 et Laboratoire de

Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL,

59045 Lille, France.

Fax: + 33 320 16 92 29, Tel.: + 33 320 16 92 18,

E-mail: [email protected]

Abbreviations: BSO, buthionine sulfoximine; CD, circular dichroı¨sm;

Cys, L-cysteine; Cys-Gly, cysteine-glycine; EI-MS, electrospray ion￾ization mass spectroscopy; c-Glu-Cys, gamma-glutamic acid-cysteine;

Gln, L-glutamine; c-Glu-Gly, gamma-glutamic acid-glycine;

GS-DCE, S-dicarboxyethyl-glutathione; GS-Me, S-methyl-glutathi￾one; GS-NO, S-nitrosoglutathione; GS-SA, glutathione sulfonic acid;

GSH-O-Et, glutathione reduced ethyl ester; GSSG, oxidized gluta￾thione; IC50, 50% inhibitory concentration; MC, mitomycin C;

Met, L-methionine; N-Ac-Cys, N-acetyl-L-cysteine.

(Received 10 March 2003, revised 05 May 2003,

accepted 12 May 2003)

Eur. J. Biochem. 270, 2848–2859 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03663.x