Following the discovery that point mutations in the kinase domain of Bcr-Abl reduce the binding affinity of imatinib and lead to drug resistance in CML patients, efforts have been directed towards the discovery of new drugs which inhibit these resistant enzymes. Two such agents are dasatinib and nilotinib. Whereas, like imatinib, x-ray analysis of crystal structures of nilotinib in complex with the Abl kinase domain reveal that this agent binds to an inactive, DFG-out conformation of the enzyme, similar studies have shown that dasatinib binds to the catalytically active state of the enzyme (Tokarski et al, Cancer Res. 2006). However, based upon in silico methods using homology models of the imatinib-binding inactive conformation of Abl some reports claim that dasatinib is capable of binding to both the active and inactive forms of the enzyme. To help address this conundrum we have employed nuclear magnetic resonance (NMR) spectroscopy to study the different conformational characteristics and dynamic changes of the Abl protein obtained upon adding ligands.

Selectively isotope labelled (15N and 13C) Abl kinase in the unphosphorylated state was produced according to published methods (Strauss et al, J. Biomolecular NMR 2005). Chemical shift data of the protein in solution were recorded by NMR spectroscopy, both with and without ligand. By measuring residual dipolar couplings (RDC) between the back-bone amide nitrogen and hydrogen atoms of amino-acid residues in the vicinity of the conserved DFG-motif (residues 370 – 410), the conformational states and equilibria of the activation loop of the kinase were established. Upon adding imatinib to the unliganded Abl, both chemical shift and RDC data show characteristic signals for residues M388, Y393 and G398, which are entirely consistent with the conformational equilibrium moving to the inactive state, in which the activation loop adopts the DFG-out conformation. In the case of nilotinib, NMR spectroscopy revealed chemical shift patterns and couplings involving the same three residues confirming that the drug binds to the same inactive conformation as imatinib, both in solution as well as in the crystalline state. In contrast, upon adding dasatinib to Abl, NMR data of the complex show distinctly different chemical shifts and RDC values, confirming that the protein assumes a different conformational state, with the activation loop adopting the active conformation, in accordance with the crystallographic evidence. Even upon adding dasatinib to a complex of imatinib with Abl in the inactive conformation, the kinase conformation changed to a state indistinguishable to that observed upon adding dasatinib to unliganded protein.

In conclusion, these studies show that the three Abl kinase inhibitors all interact with the protein in solution with the same binding modes as observed in x-ray crystallographic studies, but show no evidence for dasatinib binding to the inactive “DFG-out” conformation. This case study also demonstrates the power of NMR spectroscopy in evaluating solution structures of ligand-protein complexes. Further experiments are in progress evaluating other structurally different Abl kinase inhibitors.

Disclosures: Paul W. Manley, Sandra W. Cowan-Jacob, Gabriele Fendrich, André Strauss and Wolfgang Jahnke are full-time employees of Novartis Pharma Ltd.; Stephan Grzesiek has received Research Funding from Novartis Pharma Ltd.

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