In Vitro and In Vivo Inhibition of the T315I Mutant BCR/ABL Kinase.

The T315I point mutation situated at the ATP-binding site of BCR/ABL tyrosine kinase remains to be the major unmet medical need in treating CML patients. None of the marketed CML drugs, including Imatinib or the recently approved Dasatinib, are able to inhibit this clinically most prevalent mutant of BCR/ABL kinase. To overcome the T315I mutation-presented hurdle, a novel small molecule BCR/ABL inhibitor, TG101223, has been synthesized and characterized in vitro and in vivo. Enzymatic activity of T315I mutant kinase and proliferation of BaF3:BCR/ABL^T315I cells were inhibited with an IC₅₀ of 50 nM and EC₅₀ of 500 nM respectively. However, little effect of TG101223 was observed on the proliferation of the control BaF3 cells (IC₅₀≥5,000 nM). Consistent with these observations, 24 hour treatment with 2,000 nM TG101223 induced DNA fragmentation in BCR/ABL^T315I cells, but not in the control BaF3 cells. Furthermore, caspase-3 activation was detected in BCR/ABL^T315I cells by an in-cell caspase assay following 4 h treatment with 2,000 nM TG101223. The above results strongly suggest that TG101223 inhibits BCR/ABL^T315I-mediated cell proliferation and survival. In contrast, Imatinib did not inhibit Abl^T315I enzyme nor BCR/ABL^T315I cell proliferation. The kinase selectivity of TG101223 was determined using a commercial enzyme assay with a panel of 49 phylogentically diverse protein kinases. Only Abl, Flt3 and the mutants of those two kinases were inhibited by more than 80% with 500 nM TG101223. Importantly, TG101223 demonstrated a desirable pharmacokinetic profile in mouse (oral bioavailability: 40%, clearance: 23 mL/min/kg and T_½: 5 h). The tumorogenicity of BCR/Abl^T315I cells were confirmed by the observation that tail vein infusion with BCR/ABL^T315I cells induced splenomegaly in SCID mice and death by day 14. To assess the in vivo activities of TG101223, mice were dosed orally once with TG101223 on day 13, followed by harvesting of their spleens 7 hours after dosing for Western blot-based protein phosphorylation analysis. A significant induction in phosphorylation of both CrkL and BCR/ABL was observed in the spleens of T315I cell recipient mice, supporting the notion that splenomegaly is a result of the infiltration of proliferating BCR/ABL^T315I cells. The above induction in spleen CrkL and BCR/ABL phosphorylation was inhibited by TG101223. These data suggest that TG101223 is a promising candidate for treating the drug resistant CML patients.