Equally Potent Inhibition of c-Src and Abl by Compounds that Recognize Inactive Kinase Conformations

Markus A. Seeliger, Pratistha Ranjitkar, Corynn Kasap, Yibing Shan, David E. Shaw, Neil P. Shah, John Kuriyan, and Dustin J. Maly

Cancer Research 69, 2384, March 15, 2009 (local copy)

Abstract / Figures from the paper / Coordinates / Supplementary Information


Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia. Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2,000-fold lower. Imatinib recognition requires a specific inactive conformation of the kinase domain, in which a conserved Asp-Phe-Gly (DFG) motif is flipped with respect to the active conformation. The inability of c-Src to readily adopt this flipped DFG conformation was thought to underlie the selectivity of imatinib for Abl over c-Src. Here, we present a series of inhibitors (DSA compounds) that are based on the core scaffold of imatinib but which bind with equally high potency to c-Src and Abl. The DSA compounds bind to c-Src in the DFG-flipped conformation, as confirmed by crystal structures and kinetic analysis. The origin of the high affinity of these compounds for c-Src is suggested by the fact that they also inhibit clinically relevant Abl variants bearing mutations in a structural element, the P-loop, that normally interacts with the phosphate groups of ATP but is folded over a substructure of imatinib in Abl. Importantly, several of the DSA compounds block the growth of Ba/F3 cells harboring imatinib-resistant BCR-ABL mutants, including the Thr315Ile "gatekeeper" mutation, but do not suppress the growth of parental Ba/F3 cells. [Cancer Res 2009;69(6):2384–92

Illustrations from the paper.

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Figure 1. Structure and binding orientation of inhibitors that recognize the Abl/c-Kit–like inactive conformation of protein kinases. A, schematic representation of the subsites that are occupied by inhibitors that bind the DFG Asp-out conformation of Abl. Red, P-loop; orange, helix αC; blue, activation loop. The three distinct subregions that are occupied by imatinib (adenine pocket, specificity pocket, and exposed site) are represented with dotted circles. The letters used to label imatinib in this model directly correlate to the structure in B. B, chemical structures of imatinib, DSA1, and DSA8. All molecules are drawn in an orientation that is consistent with their mode of binding to c-Src or Abl. Red, functional groups, which are similar between the inhibitors. Each ring in these structures is labeled (rings A–F) for reference.
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Figure 2. Structurally defined conformational states of the c-Src and Abl kinase domains. Red, P-loop; orange, helix αC; blue, activation loop. State A, active c-Src kinase Thr338Ile (PDB entry 3DQW; ref. 16); state B, inactive Src/Cdk–like conformation (PDB entry 1QCF; ref. 32); state C, inactive Abl/c-Kit–like conformation (PDB entry 1OPJ; 33) with the DFG motif in the flipped conformation relative to states A and B
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Figure 3. Solvent exposure of inhibitors. Top, structures of the Abl·imatinib (A, PDB entry 1OPJ; ref. 33), c-Src·imatinib (B, PDB entry 2OIQ; ref. 3), and Src·DSA8 (C) complexes for comparison. Transparent gray, the solvent accessible surface of the proteins; red, P-loop; orange, helix αC; blue, activation loop. Each ring of imatinib and DSA8 has been labeled A to F in the structure representation (top) and the schematic drawing (bottom). Bottom, the solvent accessible area was calculated for the atoms (red) for imatinib and DSA8 in each protein complex.
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Table 1. In vitro activities of DSA1-DSA9 and imatinib against Abl, c-Src, and Hck
Note: All assays were performed in triplicate or quadruplicate.

Figure 4. Structures of wt c-Src·imatinib (A, PDB-entry 2OIQ; ref. 3), wt c-Src·DSA8 (B), and Thr338Ile c-Src·DSA1 (C). In all three complexes, c-Src is in an Abl/c-Kit–like inactive conformation with a flipped DFG motif. Top, structure of the active site region of these complexes. c-Src is shown in gray ribbons. Red, P-loop; orange, helix αC; blue, DFG motif. Parts of the protein that obscure the view of the inhibitors have been removed for clarity. Each ring in DSA1, DSA8, and imatinib has been labeled according to Fig. 1B. Bottom, schematic diagram of the binding interactions made by DSA1, DSA8, and imatinib with c-Src. Red semicircles, hydrophobic contacts; green dotted lines, H bonds with their respective lengths. The yellow arrow in the depiction of DSA1 and DSA8 indicates the rotation of ring D relative to imatinib. Schematic drawings were prepared with LigPlot (34). D, proliferation of parental Ba/F3 cells in the presence of IL-3 and Ba/F3 BCR-ABL cells and in the absence of IL-3 in response to a dose titration of DSA8. Error bars, SEM.
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Coordinates in the Protein Data Bank: 3G6H

Coordinates in the Protein Data Bank: 3G6G

Supplementary Information

Supplementary Figures