In each PFT�� supplier instance, motesanib was a more potent inhibitor of Kit autophosphorylation than imatinib. For example, motesanib inhibited the AYins503-504 mutant with an IC50 of 18 nM, whereas imatinib inhibited this mutant with an IC50 of 84 nM. Interestingly,
the IC50 values for inhibition of these Kit mutants were lower than the IC50 for inhibition of wild-type Kit by motesanib. Consistent results were obtained in a functional viability assay utilizing IL-3-independent growth of Ba/F3 cells (Figure 3C). For example, when testing the AYins503-504 mutant, the IC50 for motesanib was 11 nM versus 47 nM for imatinib. Table 2 Inhibition of the Activity of Wild-Type Kit and Primary Activating Kit Mutants by Motesanib and Imatinib* IC50 of Kit Autophosphorylation, nM IC50 of Stably Transfected Ba/F3 Cell Survival, nM KIT Genotype Motesanib Imatinib Motesanib Imatinib Wild-type 36 165 – - V560D 5 18 3 7 Δ552-559 1 5 0.4 1 AYins503-504 18 84 11 47 *In autophosphorylation experiments, means from 2 experiments are shown, with the exception of Δ552-559, which was assessed once. Viability experiments were performed once. Figure 3 Inhibition of the activity of wild-type Kit and primary activating Kit mutants by motesanib. Autophosphorylation (expressed
as a percentage of vehicle control) of wild-type Kit (panel A) and primary activating Kit mutants (panel B) was assessed in Talazoparib in vitro stably transfected Chinese hamster ovary cells treated for 2 hours with GDC-0449 in vitro single 10-fold serial dilutions of motesanib. Representative data from 1 of 2 experiments are shown. Viability (expressed as the percentage of vehicle control) of Ba/F3 cells expressing the same primary activating Kit mutants treated
for 24 hours with single 10-fold serial dilutions of motesanib was also assessed (panel C). Viability experiments were Y-27632 2HCl performed once (representative curves are shown). Activity of Motesanib against Imatinib-Resistant Kit Mutants Motesanib inhibited the activity of Kit mutants associated with secondary imatinib resistance. In Kit autophosphorylation assays, motesanib inhibited tyrosine phosphorylation of the juxtamembrane domain/kinase domain I double mutants V560D/V654A and V560D/T670I with IC50 values of 77 nM and 277 nM, respectively. Imatinib had limited activity against the V560D/V654A mutant and no activity against the V560D/T670I mutant at concentrations of up to 3000 nM (Table 3; Figure 4B). Consistent results were obtained in the Ba/F3 cells expressing the V560D/V654A and V560D/T670I mutants with motesanib IC50 values of 91 nM and 180 nM, respectively. Again, motesanib was a more potent inhibitor of these mutants than imatinib (Table 3; Figure 4C).