This effect was reversible. Following selleck compound the cessation of mote sanib treatment on day 21, the mice were depilated again Recombinant Inhibitors,Modulators,Libraries anti phosphotyrosine antibody 4G10 was added to each well and incubated at room temper ature for 1 hour. The plate was then washed 3 times with DELFIA wash buffer before 0. 01 ug of Eu N1 labeled anti mouse antibody was added to each well. The plate was again incubated at room temperature for 1 hour and then washed 3 times with DELFIA wash buffer before the signal was detected by adding DELFIA enhancement buffer to each well. Luminescence was measured using a Victor Model 1420 multilabel counter. Kit autophosphorylation at each motesanib or imatinib concentration was expressed as a percentage of the vehi cle control.

Ba F3 Functional Viability Assay The ability of Kit mutants to act as survival factors was assessed in Kit dependent Ba F3 cells. Ba F3 cells stably transfected with various KIT mutants were seeded in a 96 well tissue culture plate at a density of 5 103 cells per well. To determine IC50 values, Inhibitors,Modulators,Libraries cells were treated for 24 hours with single 10 fold serial dilutions of motesanib or imatinib starting at 3 uM. Cell viability was on day 28. There was no apparent depigmentation of regrown hair on day 35. Similar results were obtained in male mice. Characterization of Kit Mutants Figure 2 summarizes the results from the autophosphory lation experiments using CHO cells stably transfected with the wild type KIT gene or various KIT mutant genes. Tyrosine phosphorylation of wild type Kit was dose dependent, with the greatest intensity of autophos phorylation occurring after a 30 minute incubation of the cells with 300 ng mL of SCF.

In contrast, tyrosine phos phorylation of activated Kit mutants occurred in the absence of SCF with no further phosphorylation induced by treatment with SCF. Activity of Motesanib against Primary Inhibitors,Modulators,Libraries Activating Kit Mutants In CHO cells, motesanib inhibited the autophosphoryla tion of the primary activating Kit mutants V560 D, 552 559, and AYins503 504. In each instance, motesanib was a more potent inhibitor of Kit autophosphorylation than imatinib. For example, mote sanib inhibited the AYins503 504 mutant with an IC50 Inhibitors,Modulators,Libraries 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 inhibi tion of wild type Kit by motesanib.

Consistent results were obtained in a functional viability assay utilizing IL 3 independent growth of Ba F3 cells. For example, when testing the AYins503 504 mutant, the IC50 for motesanib was 11 nM Inhibitors,Modulators,Libraries versus 47 http://www.selleckchem.com/products/GDC-0449.html nM for imatinib. Activity of Motesanib against Imatinib Resistant Kit Mutants Motesanib inhibited the activity of Kit mutants associated with secondary imatinib resistance.