Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide]

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu(460), Tyr(476) Arg(525), and Asp(539) residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 Angstrom, and the thickness of the pocket is approximately 7 Angstrom. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a K-i value of 1.4 mu M, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 mu M. Similarly, LFRIA13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 mu M, The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr(476), and its substituent group is sandwiched between residues Arg(525) and Asp(539). In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp(539) and Arg(525) residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK, Even at concentrations as high as 100 mu g/ml (similar to 278 mu M), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase, In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis, To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.