Targeted inhibition of Gatekeeper variant “T315I” of BCR-ABL by a purine based ATP-competitive inhibitor

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In patients with chronic myeloid leukemia (CML), kinase domain mutations account for imatinib resistance in about 50-90% of cases. Mutations cause either a direct steric hindrance to drug binding or a conformational change that favors kinase activation, which therefore precludes imatinib binding. We have analyzed two dual Src-Abl kinase inhibitors, AP23464 and PD166326, for structure activity relationship (SAR). Both of these compounds have effectively suppressed the cell growth of native and imatinib resistant variants of BCR-ABL though with varying degree of sensitivity, except the gatekeeper residue (T315I). Imatinib resistance due to T315I substitutions represents 25-30 % of total resistance and is not inhibited by any BCR-ABL inhibitors. Earlier, we have demonstrated that the combination of different conformation specific inhibitors such as AP23464 (active conformation), PD166326 (intermediate conformation) and Imatinib (inactive conformation) essentially captures all resistance conferring mutations with notable exception of T315I. Based on our in vitro and in silico analysis we modified the AP23464 to develop AP23846 and AP23994 in order to inhibit T315I variants. Both of these compounds are effective in inhibiting the tyrosine kinase activity of native and T315I variants of BCR-ABL within 400-600 nM range. However, it also inhibited the parental cells, most likely by inhibiting some unknown target. Later, we redesigned these molecules to create AP24163 in order to have selectivity for T315I variant and lack of toxicity on parental cells. Indeed AP24163 inhibits the native and T315I variant of BCR-ABL kinase activity with IC50 values of 5nM and 350 nM, respectively. While the proliferation of parental BAF3 cells was not affected up to 5000 nM. This in-vitro study is a paradigm applicable to a list of target-directed agents and means to understand structure activity relationship in order to develop more potent inhibitors to combat resistance.