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Characterization of compound 584, an Abl kinase inhibitor with lasting effects

¹ Department of Clinical Medicine, University of Milano-Bicocca, S. Gerardo Hospital, Monza, Italy
² Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland
³ Department of Environmental Sciences, University of Ca’ Foscari, Venezia, Italy
⁴ Department of Experimental Oncology, National Cancer Institute, Milan, Italy
⁵ Department of Biological Chemistry University of Padova, Padova, Italy
⁶ ProQinase GmbH, Freiburg, Germany
⁷ Department of Oncology, McGill University, Montreal, Quebec, Canada

Correspondence: Leonardo Scapozza, PhD, School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, 1211 Genève 4, Switzerland. E-mail: leonardo.scapozza{at}pharm.unige.ch

ABSTRACT

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584).

Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor.

Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC₅₀: 8 nM) and in cell-based assays (IC₅₀: 0.1–10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC₅₀: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib.

Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

Key words: Abl, tyrosine kinase inhibitor, off-rate, imatinib analog.