Sentization of BCL-2 Overexpressing Cells to Apoptosis by Primidine Derivative

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The Mitochondrial Effects of Small Organic Ligands of BCL-2

SENSITIZATION OF BCL-2-OVEREXPRESSING CELLS TO APOPTOSIS BY A

PYRIMIDINE-2,4,6-TRIONE DERIVATIVE*

Eva Milanesi12, Paola Costantini13, Alberto Gambalunga, Raffaele

Colonna, Valeria Petronilli, Cabrelle¶, Gianpietro Semenzato¶,

M. Cesura||4, Emmanuel Pinard||, and Paolo Bernardi¶5

From the Department of Biomedical Sciences and Consiglio Nazionale

delle Ricerche Institute of Neurosciences, University of Padova,

Viale Giuseppe Colombo 3, I-35121 Padova, Italy, the Department of

Clinical and Experimental Medicine, University of Padova, Via

Giustiniani 2, I-35128 Padova, Italy, the ¶Venetian Institute of

Molecular Medicine, Via Orus, I-35129 Padova, Italy, and the ||Pharma

Division, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland

We have investigated the mitochondrial effects of BH3I-2',

Chelerythrine, and HA14-1, small organic molecules that share the

ability to bind the BH3 domain of BCL-2. All compounds displayed a

biphasic effect on mitochondrial respiration with uncoupling at low

concentrations and respiratory inhibition at higher concentrations,

the relative uncoupling potency being BH3I-2' (half-maximal

uncoupling at about 80 nM) > Chelerythrine (half-maximal uncoupling

at about 2 µM) > HA14-1 (half-maximal uncoupling at about 20 µM). At

concentrations lower than required for uncoupling all compounds

sensitized the permeability transition pore (PTP) to opening both in

isolated mitochondria and intact cells. To assess whether the effects

on BCL-2 binding, PTP induction and respiration could be due to

different structural determinants we have tested a set of HA14-1

analogs from the Hoffmann-La Roche chemical library. We have

identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-

diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a

molecule devoid of effects on respiration that is able to induce PTP

opening, to disrupt the BCL-2/BAX interactions in situ and to

activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized

the antiapoptotic activity of overexpressed BCL-2 toward

staurosporine and sensitized BCL-2-expressing cells from leukemic

patients to the killing effects of staurosporine, chlorambucil, and

fludarabine. These results provide a proof of principle that the

potentially toxic effects of BCL-2 ligands on mitochondrial

respiration are not essential for their antiapoptotic activity and

represent an important step forward in the development of tumor-

selective drugs acting on BCL-2.

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Received for publication, December 23, 2005 , and in revised form,

February 6, 2006.

* This work was supported in part by grants from the Associazione

Italiana per la Ricerca sul Cancro and the Regione del Veneto (to P.

B. and G. S.) and from the Ministry for the University and Research

(to P. B.). The costs of publication of this article were defrayed in

part by the payment of page charges. This article must therefore be

hereby marked " advertisement " in accordance with 18 U.S.C. Section

1734 solely to indicate this fact.

1 These authors contributed equally to this work.

2 Present address: Congenia s.r.l., Via dei Bossi 2a, 20121 Milano,

Italy.

3 Present address: Dept. of Biology, University of Padova, Viale

Giuseppe Colombo 3, I-35121 Padova, Italy.

4 Present address: Evotec NeuroSciences, August-Forel Strasse 1, CH-

8008 Zürich, Switzerland.

5 To whom correspondence should be addressed: Dept. of Biomedical

Sciences, University of Padova, Viale Giuseppe Colombo 3, I-35121

Padova, Italy. Fax: 39-049-827-6361; E-mail: bernardi@....