A Study of the Binding Mode and the In Vitro Activity of the Protein Tyrosine Kinase Inhibitor SKI-606 in the BCR-ABL Positive Cells.

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) is often caused by the selection of leukemic clones harboring mutations that destabilize the inactive conformation of BCR-ABL, to which IM preferentially binds, shifting the equilibrium toward the active kinase conformation. Hence the need for second -generation kinase inhibitors with a greater flexibility in binding to different BCR-ABL conformations. The 4-anilino-3 quinolinecarbonitrile SKI-606 is a novel Src and ABL kinase inhibitor. We report here that SKI-606 is a potent and antiproliferative agent when tested in K562 cell line and CD34⁺ cells from patients with chronic myeloid leukemia (CML) blast crisis. In K562 cells, SKI-606 treatment caused a dose-dependent decrease in cell viability accompanied by accumulation in subG₁ phase, an effect not observed in BCR-ABL-negative HL-60 cells. Treatment of K562 with 100 nM SKI-606 for 24 hours caused a complete dephosphorylation of Lyn, Stat5 and Bcl-xl, while AKT and Bad phosphorylation was only diminished. Unexpectedly, the phosphorylation of BCR-ABL was less affected. SKI-606 treatment caused a shift to the subG₁ phase also of CD34⁺ cells isolated from both IM-sensitive and resistant patients, the latter harboring the BCR-ABL mutations F359V, Y253H, E255V and E255K. The inhibitory concentration 50% (IC50) was 100 nM SKI-606 for Y253H, E255V, E255K and 1000nM for F359V. Cytofluorimetric analysis of cells from IM- sensitive CML patients indicated an accumulation in subG₁ phase following treatment with SKI-606 alone or in combination with IM. Because the crystal structure of the BCR-ABL kinase domain in complex with SKI-606 has not yet been determined and the mode of binding of this inhibitor is unknown, we first used a molecular docking approach to determine SKI-606 binding mode to wild type (wt) form of the BCR-ABL kinase. We found that the interaction between SKI-606 and BCR-ABL was more stable when the activation loop was in the inactive conformation. Moreover, we found that SKI-606 retained the ability of efficiently binding all the above mentioned BCR-ABL variants, but not the T315I. Finally, we identified six BCR-ABL residues located around SKI-606 that, if mutated, could potentially be able to interfere with the SKI-606/BCR-ABL interaction: the charged residues K271, D381 and H361; the hydrophobic/aliphatic residues V299, A380 and M318.

These data help refining the use of SKI-606 for treatment of BCR-ABL positive leukemias.