Identification and characterization of inhibitors of cytoplasmic 5 0 -nucleotidase cN-II issued from virtual screening Lars Petter Jordheim a,b,c, *, Zsuzsanna Marton d , Moez Rhimi e , Emeline Cros-Perrial a,b,c , Corinne Lionne d , Suzanne Peyrottes f , Charles Dumontet a,b,c , Nushin Aghajari e , Laurent Chaloin d a Universite ´ de Lyon, F-69000 Lyon, France b INSERM U1052, Centre de Recherche en Cance ´rologie de Lyon, F-69000 Lyon, France c CNRS UMR 5286, Centre de Recherche en Cance ´rologie de Lyon, F-69000 Lyon, France d Centre d’e ´tudes d’agents Pathoge `nes et Biotechnologies pour la Sante ´ (CPBS), UMR 5236, CNRS – Universite ´s Montpellier 1 et 2, 1919 route de Mende, 34293 Montpellier cedex 5, France e Laboratory for BioCrystallography and Structural Biology of Therapeutic Targets, ‘‘Bases Mole ´culaires et Structurales des Syste `mes Infectieux’’ UMR 5086 CNRS/Universite ´ de Lyon 1, Institut de Biologie et Chimie des Prote ´ines FR3302, 7 Passage du Vercors, F-69367 Lyon cedex 07, France f Institut des Biomole ´cules Max Mousseron (IBMM), UMR 5247, CNRS – Universite ´s Montpellier 1 et 2, Universite ´ Montpellier 2, Place Euge `ne Bataillon, 34095 Montpellier cedex 5, France 1. Introduction The cytoplasmic 5 0 -nucleotidase cN-II is one of seven 5 0 - nucleotidases found in mammals [1] and belonging to the haloacid dehalogenase superfamily. cN-II has a substrate preference for purine 5 0 -monophosphates, especially IMP and GMP [2], and is involved in the maintenance of cellular nucleotide pools through the regulation cycle made of nucleoside kinases and 5 0 -nucleoti- dases [3]. Work over the last 15 years has allowed gaining insights into the regulation of cN-II activity, including the formation of tetramers, the presence and identification of allosteric regulation as well as for the phosphotransferase activity [4,5]. Understanding the activity and the regulation of cN-II has become easier since the publication of crystal structures of the enzyme both in its apo form and in the presence of substrates and regulators [6,7]. In addition to its physiological role in human cells, cN-II is correlated to the clinical outcome of cancer patients treated with cytotoxic nucleoside analogs. In particular, patients with acute myeloid leukemia and low expression level of cN-II have a better overall survival after treatment with cytarabine than patients with high expression level [8]. Since this observation in 2001, we and others have confirmed the role of cN-II as a predictive factor for the outcome of similar patient groups [9,10]. Mazzon et al. observed that cN-II does not, or only weakly, dephosphorylate monopho- sphorylated cytarabine, gemcitabine or cladribine in vitro, whereas we demonstrated that fludarabine monophosphate is a substrate Biochemical Pharmacology 85 (2013) 497–506 A R T I C L E I N F O Article history: Received 23 August 2012 Accepted 29 November 2012 Available online 7 December 2012 Keywords: cN-II Virtual screening Enzymatic assay Inhibitor Crystallography A B S T R A C T Clinical and preclinical observations have lead to the hypothesis that 5 0 -nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs. To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells. The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a K i of 2.0 mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9 A ˚ and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5 mM cladribine, 0.05 mM clofarabine or 30 mM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine. This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics. ß 2012 Elsevier Inc. All rights reserved. * Corresponding author at: Equipe Anticorps-Anticancer, Centre de Recherche en Cance ´ rologie de Lyon, INSERM U1052 – CNRS UMR 5286, Faculte ´ Rockefeller, 8 avenue Rockefeller, 69008 Lyon, France. Tel.: +33 478777128; fax: +33 478777088. E-mail address: lars-petter.jordheim@univ-lyon1.fr (L.P. Jordheim). Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.11.024