The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 <30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.

SUMMARY In an effort to find new pharmacological modalities to overcome resistance to ATP-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry we demonstrate that GNF-2 binds to the myristate binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analog of GNF-2 having improved pharmacokinetic properties, when utilized in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I Bcr-Abl and displayed in vivo efficacy against the recalcitrant T315I Bcr-Abl mutant in a murine bone-marrow transplantation model. These results demonstrate that therapeutically relevant inhibition of Bcr-Abl activity can be achieved using inhibitors that bind to the myristate binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

In this article (Cancer Cell 7,, the name of one of the authors was listed incorrectly. The author incorrectly listed as "Azam Mohammed" is actually named Mohammad Azam.

The regulation of the activity of Abl and Src family tyrosine kinases is mediated by intramolecular interactions between the SH3, SH2, and kinase (SH1) domains. We have determined the crystal structure of an unphosphorylated form of c-Src in which the SH2 domain is not bound to the C-terminal tail. This results in an open structure where the kinase domain adopts an active conformation and the C terminus binds within a hydrophobic pocket in the C-terminal lobe. NMR binding studies support the hypothesis that an N-terminal myristate could bind in this pocket, as observed for Abl, suggesting that c-Src may also be regulated by myristate binding. In addition, the structure contains a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). This structure reveals why the drug shows a low affinity for active kinase conformations, contributing to its excellent kinase selectivity profile.

A case study showing how the determination of multiple cocrystal structures of the protein tyrosine kinase c-Abl was used to support drug discovery, resulting in a compound effective in the treatment of chronic myelogenous leukaemia.

Current structural understanding of kinases is largely based on x-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. ABL kinase is an important drug target in the treatment of chronic myelogenous leukemia. Here, we present the first characterization of ABL kinase in complex with three clinical inhibitors (imatinib, nilotinib, and dasatinib) by modern solution NMR techniques. Structural and dynamical results were derived from complete backbone resonance assignments, experimental residual dipolar couplings, and 15 N relaxation data. Residual dipolar coupling data on the imatinib and nilotinib complexes show that the activation loop adopts the inactive conformation, whereas the dasatinib complex preserves the active conformation, which does not support contrary predictions based upon molecular modeling. Nanosecond as well as microsecond dynamics can be detected for certain residues in the activation loop in the inactive and active conformation complexes.Protein kinases play critical roles in intracellular signal transduction pathways, deregulation of which can lead to a variety of pathological states and diseases such as cancer. These enzymes are therefore tightly regulated with multiple layers of control, including phosphorylation, myristoylation, and interaction with SH2 3 and SH3 or other regulatory domains. Modulation of kinase activity by therapeutic agents is a clinically validated concept, with many kinases considered to be attractive drug targets. ABL kinase is such a target because the expression of the BCR-ABL fusion protein (caused by unfaithful repair of DNA strand breaks in bone marrow hematopoietic stem cells and subsequent t(9,22) chromosome translocation) leads to life-threatening chronic myelogenous leukemia (1, 2). In BCR-ABL, the breakpoint cluster region BCR protein replaces the N-terminal autoregulatory domain of the Abelson ABL protein to give a constitutively activated tyrosine kinase, which deregulates signal transduction pathways, causing uncontrolled proliferation and impaired differentiation of progenitor cells.X-ray crystallography has revealed various active and inactive conformational states of kinases, which are implicated in their regulation and modulation by inhibitors (3). The active states are characterized by certain conformations of the activation loop, phosphate-binding loop (P-loop), and helix C, which orient the catalytic machinery to phosphorylate substrates; in the inactive states, one or more of these elements are in different conformations, such that substrate binding and/or catalysis cannot occur. An important determinant is the orientation of the conserved Asp-Phe-Gly motif within the activation loop. For efficient catalysis, this motif adopts a "DFG-in" conformation. In contrast, the "DFG-out" conformation has this motif displaced from the orientation needed for binding the substrate ATP to phosphorylate and activate downstream signaling proteins. Such a DFG-out conformation has been ...

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