The kinase inhibitor imatinib (Gleevec®, Novartis) has enabled highly effective therapy for chronic myeloid leukemia (CML) (1
). The clinical success of imatinib is due partly to its high affinity for the tyrosine kinases BCR-ABL, c-Abl, c-Kit and PDGFR, combined with much reduced affinity for most other kinase targets, including the Src-family kinases that are most closely related to Abl. Simple comparisons of sequence and structure do not provide an explanation for why imatinib is inactive against c-Src. The structure of the c-Src•imatinib complex closely resembles that of the Abl•imatinib complex, despite the 2300-fold difference in affinity between c-Src and Abl kinase domains for imatinib (3
Imatinib is derived from a 2-phenylaminopyrimidine scaffold (rings C and D, ) that is substituted with a pyridine (ring B) and a benzamide-piperazine (rings E and F). Imatinib is bound deeply in the cleft between the N- and C-lobes of the kinase via
three binding sites (). The pyridine and pyrimidine moieties (rings B and C) bind to the site normally occupied by the adenine group of ATP (adenine pocket, ). The meta
-diaminophenyl and benzamide group (rings D and E) are accommodated within a pocket that is enlarged greatly by adoption of an inactive conformation by the kinase domain (specificity pocket, ). The methyl-piperazine group (ring F) binds to a hydrophobic patch on the surface of the kinase domain (exposed site, ). Imatinib makes only four hydrogen bonds with Abl and the majority of interactions are mediated through van der Waals contacts, with the burial of ~1300 Å2
of surface area between the drug and the protein (4
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. The (more ...)
The adoption of a specific inactive conformation by the kinase domain of Abl is key to the recognition of imatinib (5
). Three distinct conformations of the kinase domains of c-Src and Abl have been described structurally: an active conformation (, state A), a c-Src/Cdk-like inactive conformation (, state B), which is similar to the inactive conformations of Src-family kinases and cyclin dependent kinases (Cdks) (6
), and an Abl/c-Kit-like inactive conformation (, state C) (5
), which was first observed in the imatinib-complexes of those kinases. The conformation of the conserved Asp-Phe-Gly (DFG) motif at the beginning of the activation loop is a key feature that distinguishes between these conformations. In the active conformation (, state A) the aspartate sidechain faces into the ATP-binding pocket and coordinates a Mg2+
ion, and the phenylalanine sidechain is rotated out of the ATP binding pocket (DFG-Asp in). In the Abl/c-Kit-like inactive conformation (, state C) the DFG motif is flipped, the phenylalanine sidechain occupies the ATP binding pocket and the aspartate sidechain faces away from the active site (DFG-Asp out). In the c-Src/Cdk-like inactive conformation (, state B) the DFG conformation is intermediate between DFG-Asp out and DFG-Asp in.
Figure 2 Structurally defined conformational states of the c-Src and Abl kinase domains. The P-loop is colored in red, helix αC is colored in orange and the activation loop is colored in blue. State A, active c-Src kinase Thr338Ile (PDB-entry 3DQW, (16 (more ...)
It has been assumed that the low affinity of imatinib for c-Src is a consequence of an inability of c-Src to adopt the Abl/c-Kit-like inactive conformation. That the explanation is not so simple is indicated by the crystal structures of imatinib bound to c-Src (3
) and the Src-family kinase Lck (11
). These crystal structures showed that when c-Src and Lck bind to imatinib they adopt the Abl/c-Kit-like inactive conformation, with the DFG motif flipped into the DFG–Asp out conformation.
The difference in imatinib affinity for c-Src and Abl cannot be explained by the loss of hydrogen bonds or by steric occlusion. In terms of contacts with the drug, the differences between the c-Src and Abl complexes are limited to a strand-loop-strand motif called the P-loop, which interacts with the phosphate groups and the ribose group of ATP in active kinases. In the Abl•imatinib complex, the P-loop is folded over the pyridine and pyrimidine groups (rings B and C, ) of imatinib, shielding them from water (). Mutations that confer resistance to imatinib occur with the highest frequency in the P-loop of the Abl kinase domain (12
). It has been speculated that a majority of these mutations confer resistance by destabilizing the particular kinked conformation of the P-loop that Abl adopts when bound to imatinib. In contrast, the P-loop is undistorted in the c-Src•imatinib complex ().
Figure 3 Solvent exposure of inhibitors. Top panel. The structures of the Abl•imatinib (A, PDB-entry 1OPJ) (33), c-Src•imatinib (B, PDB-entry 2OIQ) (3) and Src•DSA8 (C) complexes are shown for comparison. The solvent accessible surface (more ...)
The functional relevance of these differences in the P-loop conformation was unclear for the following reasons (i) the P-loop is extended in the high affinity c-Kit•imatinib complex (9
), as it is in c-Src (3
), (ii) the P-loop appears to make non-specific hydrophobic contacts with imatinib and (iii) replacement of the P-loop in c-Src by the corresponding residues in Abl did not increase the affinity for imatinib (3
). Also, Lck has a very similar P-loop sequence, but has a higher affinity for imatinib than does c-Src (3
). For these reasons, we have speculated previously that the Abl/c-Kit-like conformation may be associated with a relatively high free energy in c-Src, and that the low affinity of imatinib for c-Src might result from the energy required to convert the c-Src/Cdk-like inactive conformation to the Abl/c-Kit-like inactive conformation (3
). Similarly, the noted inability to confer imatinib sensitivity on c-Src kinase domain by limited sequence swaps implies that the effect arises due to a distributed set of differences between Src and Lck (3
). These differences include the interactions seen in Abl between the N-lobe, the C-lobe and the activation loop that stabilize the kinked conformation of the P-loop. The relative stability of the inactive Src/Cdk-like inactive conformation may also be a contributing factor. For example, the c-Src/Cdk-like inactive conformation may be more stable for Src than for Lck. Accordingly, mutations in the c-Src kinase domain, designed to destabilize the inactive conformation, increased the affinity of this enzyme for imatinib 16-fold. The observed Ki
-value of 2 μM for these c-Src mutants is close to the 0.4 μM Ki
-value of Lck kinase domain (3
About 35% of all CML patients undergoing imatinib treatment accumulate mutations in the Abl kinase domain that render the kinase resistant to imatinib (12
). The energetic balance between different conformations of the kinase domain is relevant for understanding the mechanisms by which mutations in the kinase domain of Abl reduce imatinib affinity. Even in cases where mutations result in steric blockage of imatinib binding, this conformational balance may be relevant. For example, a mutation (Thr315Ile) at the gatekeeper position of Abl (so named because of its location at the junction of the adenine pocket and the specificity pocket) leads to activation of the mutant protein in vivo
). This gatekeeper mutation is of particular clinical interest because it results in resistance to second generation Abl kinase inhibitors such as dasatinib and nilotinib (17
). In addition, mutation at the equivalent position in c-Kit and PDGFR kinase causes resistance to imatinib (20
). Mutation at the gatekeeper residue in EGFR kinase causes clinical resistance to gefitinib and erlotinib by increasing the affinity for ATP (22
In this study we have generated a series of inhibitors (DSA1-DSA9) that are based on the central chemical scaffold of imatinib () and that are designed to bind to kinases with a flipped DFG conformation. We find that these derivatives, unlike imatinib, are equipotent inhibitors of both c-Src and Abl, with inhibitory constants in the nanomolar range. Crystal structures of the c-Src kinase domain bound to two of these inhibitors (DSA1 and DSA8) reveal that c-Src readily adopts the DFG-Asp out conformation, which is supported by kinetic data.
Clearly, DFG-flips are not hindered in the c-Src kinase domain. Instead, our results indicate that the different accommodation of the hydrophobic face of the pyridine ring (ring B, ) of imatinib by the P-loop of c-Src and Abl are the key to understanding the selectivity of this drug ().