Extended kinase profiling of the Bcr-Abl inhibitor nilotinib
Authors: Paul Manley, Josef Bruggen, Doriano Fabbro, Georg Martiny-Baron, Thomas Meyer
Journal: Cancer Research (2007)

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

3249 

The new Bcr-Abl tyrosine kinase inhibitors nilotinib and dasatinib, developed for the treatment of imatinib-resistant chronic myelogenous leukemia (CML), show similar efficacy in patients but different safety profiles. Nilotinib is a potent inhibitor of wild-type and mutant Bcr-Abl, as well as c-Kit and PDGFR (Br. J. Cancer 2006;94:1765), whereas dasatinib in addition to inhibiting Bcr-Abl, c-Kit and PDGFR, potently inhibits Src family kinases and the EphA2 receptor kinase. Here we report the activity of nilotinib against a number of other protein kinases. Enzyme activities were measured by mixing test compound with peptidic substrate, ATP and [γ33P]ATP, followed by the addition of respective enzyme. Enzymatic reactions were stopped by adding EDTA and the incorporation of 33P into the polymeric substrate was quantified by either the flash-plate method (c-Src) or the filter binding mode (all other kinases). The data below represent mean IC50 values ± SEM (nM; n≥3). Nilotinib displayed little inhibitory activity against c-Src (4600±520), Lyn (2700±460), Hck (7500±830), Lck (5200±610), Fyn (>10000) or Fgr (>10000). In comparison, dasatinib potently inhibited c-Src (12±4), Lyn (3±1), Hck (17±3), Lck (22±7), Fyn (5±1) and Fgr (3±1). Nilotinib also showed very weak activity against the receptor tyrosine kinases Tie-2 (3300±850), HER-2 (5800±3600), VEGFR-2 (5300±800) and the serine/threonine specific protein kinases C-Raf (1200±670), B-RafV600E (5100±860) and p38 (1700±440), but had no significant activity (IC50 >10 µM) against 33 other tyrosine- and serine/threonine-specific protein kinases. However, using a capture ELISA with A375 cells stably transfected with full-length EphB4, nilotinib inhibited EphB4 autophosphorylation (mean IC50 240 nM).

Although the EphB4 receptor tyrosine kinase is found on venous endothelial cells and the exact expression pattern in adults remains to be elucidated, it has been proposed as a target to inhibit tumor angiogenesis (Neoplasia 2006;6:248). Consequently we examined the inhibition of EphB4 activity in vivo . Following single oral administration to mice (50 mg/kg), nilotinib inhibited the kinase , with the amount of phosphorylated EphB4 in lung tissue being significantly reduced 2 hr post administration, when plasma and lung tissue concentrations were 40-50 nmol/mL and 40-50 nmol/g respectively. For comparison, in murine models of CML nilotinib displays good efficacy with a 50 mg/kg q24h dose leading to tumor stasis (Proc. Am. Assoc. Cancer Res, 2006;47:61). This data indicates that the efficacy of nilotinib in imatinib-resistant CML patients is due to targeted inhibition of Bcr-Abl and that Src family kinases play no role in the pharmacology of this compound. The activity against c-Kit and PDGFR has been shown to have therapeutic utility in the treatment of GIST and HES, however at present it is unclear whether or not the inhibition of EphB4 has any physiological relevance in nilotinib treated subjects.