Systematic Profiling and Novel Targets of the Bcr-Abl Kinase Inhibitors Imatinib, Nilotinib and Dasatinib.

The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia (CML). Since imatinib resistance has become a major clinical problem in recent years, two second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL and targeting imatinib-resistant CML clones were developed. To predict potential side effects and novel medical uses we generated comprehensive drug-protein interaction profiles for all three drugs. We have used an unbiased chemical proteomics approach to detect drug-binding proteins directly from lysates of CML cells. Our studies yielded four major findings:

• The interaction profiles of the three drugs displayed strong differences and only a small overlap covering the ABL kinases.

• Dasatinib bound in excess of thirty Tyr− and Ser−/Thr−kinases, including major regulators of the immune system suggesting that dasatinib might have a particular impact on immune function.

• Despite the high specificity of nilotinib the receptor tyrosine kinase DDR1 was identified and validated as an additional major target.

• The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations representing the first non-kinase target of these drugs.

In addition, we have concentrated on the promiscuous binding profile of dasatinib: Besides Abl and Src kinases, we have identified the Tec kinases Btk and Tec, but not Itk, as major binders of dasatinib. The kinase activity of Btk and Tec, but not of Itk, was inhibited by nanomolar concentrations of dasatinib in vitro and in cultured cells. We identified the gatekeeper residue as the critical determinant of dasatinib susceptibility. Mutation of Thr-474 in Btk to Ile and Thr-442 in Tec to Ile conferred resistance to dasatinib, while mutation of the corresponding residue in Itk (Phe-435) to Thr sensitized the otherwise insensitive Itk to dasatinib. The configuration of this residue may be a predictor for dasatinib sensitivity across the kinome. Analysis of mast cells derived from Btk-deficient mice suggested that inhibition of Btk by dasatinib may be responsible for the observed reduction in histamine release upon dasatinib treatment. Furthermore, dasatinib inhibited histamine release in primary human basophils and secretion of pro-inflammatory cytokines in immune cells. The observed inhibition of Tec kinases by dasatinib predicts immunosuppressive (side) effects of this drug and may offer new therapeutic opportunities for inflammatory and immunological disorders.