Abstract
Imatinib mesylate (Gleevec®, Novartis Pharma AG), an inhibitor of the c-Abl/Bcr-Abl, c-Kit, and PDGFR kinases, is an effective therapy for CML, GIST and HES. However, advanced stage CML and Ph+ ALL patients frequently relapse due to the development of resistance. In up to 50% of cases, imatinib resistance is the result of the clonal expansion of cells expressing Bcr-Abl kinase domain mutants and more than 30 such mutations have been detected. Different mutations display varying degrees of resistance, and although some are sensitive to increased concentrations of imatinib, new Abl kinase inhibitors with higher potency against wild-type and imatinib-resistant mutants of Bcr-Abl could have substantial clinical utility. Rational drug design based on the structural information obtained from the crystal structure of the Abl-imatinib complex and medicinal chemistry optimization of drug-like properties, resulted in the discovery of AMN107. The potencies of AMN107 and imatinib were compared with cellular assays using CML lines (K562 and KU812F) and Ba/F3 cells expressing either ‘wt’- or imatinib-resistant mutants of Bcr-Abl. AMN107 effectively reduced the cellular Bcr-Abl autophosphorylation in K562, KU812F and Ba/F3 Bcr-Abl cells with IC50s of 42, 60 and 23 nM, respectively (Gleevec: 470, 399 and 231 nM) The proliferation of these cells was inhibited with IC50s of 11, 8 and 23 nM (Gleevec: 272, 80 and 634 nM). Autophosphorylation of the Bcr-Abl mutants M351T, F317L and E255V was reduced by AMN107 with IC50s of 33, 43 and 245 nM (Gleevec: 595, 818 and 6499 nM), resulting in inhibition of cell proliferation with IC50s of 30, 77 and 684 nM (Gleevec: 1285, 1583 and 6294 nM). No effect could be observed below 8 μM against the T315I and G250E bcr-abl mutants. Similar to imatinib, AMN107 inhibited the cellular tyrosine kinase activity of PDGFR-β (A31 cells) and c-Kit (GIST882 cells), with IC50s of 85 and 192 nM (Gleevec: 74 and 96 nM). Control (Ba/F3) cell proliferation was unaffected by 10 μM AMN107, and added IL-3 abrogated the antiproliferative effects on Bcr-Abl expressing Ba/F3 cells. Furthermore, AMN107 (< 10 μM), did not effect unrelated kinases, e.g. c-erbB2 (BT474), Ins-R (A14) and IGF-1R (NWT-21), in cellular assays, consistent with its specificity and lack of general cytotoxicity. Based on these in vitro assays and preclinical in vivo and pharmacokinetic data to be presented separately, AMN107 represents a promising new tyrosine kinase inhibitor for testing in human clinical trials.
Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression
