Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations

doi:10.1158/0008-5472.CAN-08-3953

Cancer Res. Author manuscript; available in PMC 2010 March 15.

Published in final edited form as:

Cancer Res. 2009 March 15; 69(6): 2384–2392.

Published online 2009 March 10. doi: 10.1158/0008-5472.CAN-08-3953

PMCID: PMC2678021

NIHMSID: NIHMS93695

Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations

Markus A. Seeliger,^1,² Pratistha Ranjitkar,³ Corynn Kasap,⁴ Yibing Shan,⁵ David E. Shaw,^5,⁶ Neil P. Shah,⁴ John Kuriyan,^1,^2,⁷^* and Dustin J. Maly³^*

¹ Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

² Department of Molecular and Cell Biology and Department of Chemistry, University of California, Berkeley, California 94720, USA

³ Department of Chemistry, University of Washington, Seattle, Washington 98195, USA

⁴ Division of Hematology/Oncology, Department of Medicine, UCSF School of Medicine, San Francisco, CA 94143, USA

⁵ D. E. Shaw Research, New York, NY 10036, USA

⁶ Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032, USA

⁷ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720, USA

^* Corresponding authors: Dustin Maly (Email: maly/at/chem.washington.edu, phone: (206) 543-1653) or John Kuriyan (Email: kuriyan/at/berkeley.edu, phone (510) 643-0137, fax (510) 643-2352)

M. A. Seeliger and P. Ranjitkar contributed equally to this work.

The authors declare no competing financial interest.

The publisher's final edited version of this article is available free at Cancer Res

See other articles in PMC that cite the published article.

Abstract

Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia (CML). Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2000-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, but not in c-Src. 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.

Keywords: Leukemia, Abl, imatinib, resistance mutations, structure

Introduction

The kinase inhibitor imatinib (Gleevec®, Novartis) has enabled highly effective therapy for chronic myeloid leukemia (CML) (1, 2). 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, Figure 1B) 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 (Figure 1A). The pyridine and pyrimidine moieties (rings B and C) bind to the site normally occupied by the adenine group of ATP (adenine pocket, Figure 1A). 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, Figure 1A). The methyl-piperazine group (ring F) binds to a hydrophobic patch on the surface of the kinase domain (exposed site, Figure 1A). 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 (Figure 2, state A), a c-Src/Cdk-like inactive conformation (Figure 2, state B), which is similar to the inactive conformations of Src-family kinases and cyclin dependent kinases (Cdks) (6–8), and an Abl/c-Kit-like inactive conformation (Figure 2, state C) (5, 9, 10), 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 (Figure 2, 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 (Figure 2, 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 (Figure 2, 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, Figure 1B) of imatinib, shielding them from water (Figure 3A). Mutations that confer resistance to imatinib occur with the highest frequency in the P-loop of the Abl kinase domain (12–14). 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).

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 K_i-value of 2 μM for these c-Src mutants is close to the 0.4 μM K_i-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, 15). 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 (16). This gatekeeper mutation is of particular clinical interest because it results in resistance to second generation Abl kinase inhibitors such as dasatinib and nilotinib (17–19). In addition, mutation at the equivalent position in c-Kit and PDGFR kinase causes resistance to imatinib (20, 21). Mutation at the gatekeeper residue in EGFR kinase causes clinical resistance to gefitinib and erlotinib by increasing the affinity for ATP (22, 23).

In this study we have generated a series of inhibitors (DSA1-DSA9) that are based on the central chemical scaffold of imatinib (Figure 1B) 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, Figure 1B) of imatinib by the P-loop of c-Src and Abl are the key to understanding the selectivity of this drug (Figure 3).

Materials and Methods

Protein purification and kinetic assays

All kinases were expressed and purified as previously described (24). Stopped flow kinetics of drug binding were performed as previously described (25).

Crystallization and structure determination

Crystals of the Src•DSA complexes grew in hanging drops (1 μL of protein + 1 μL of mother liquor) at 20 °C overnight (Table S1). Crystals were cryoprotected in mother liquor plus 20 % glycerol, frozen and stored in liquid nitrogen. The structures were solved by molecular replacement in Phaser (26) built in Coot (27), and refined with refmac 5.4 (28)

Cell growth viability studies of Ba/F3 cells harboring BCR-ABL kinase domain mutations

Exponentially growing Ba/F3 cells (1 × 10⁵) were plated in media containing IL-3 (parental Ba/F3 cells) or lacking IL-3 (BCR-ABL-expressing Ba/F3 cells) in the presence of varying concentrations of DSA7 or DSA8 for 48 hours. The number of trypan blue-excluding cells were determined using an automated viable cell counter as previously described and normalized to the mock-treated sample (29). Experiments were performed in triplicate. BCR-ABL amino acid substitutions were numbered according to the Abl type 1a convention.

Results and Discussion

Equipotent inhibitors of the tyrosine kinases c-Src and Abl that are designed to recognize flipped DFG motifs

We were intrigued by a recent report describing a series of inhibitors that bind to the DFG-Asp out conformation of the receptor tyrosine kinase Tie-2 (30). Lead compounds from this inhibitor series were reported to be potent inhibitors of the kinases p38α, c-Kit, Lck, Lyn and c-Src (IC₅₀ ≤ 25 nM) (30). We were interested in determining the conformation that c-Src adopts when bound to inhibitors based on these scaffolds. To this end, we synthesized pyridinyl triazine DSA1 (Figure 1B, Table 1). DSA1 contains many of the same functional groups that are exploited by imatinib to bind to the DFG-Asp out conformation of Abl. The exocyclic nitrogen from the trimethoxyaniline ring (ring A) and one of the nitrogens from the triazine ring (ring B) are expected to form hydrogen bonds with the hinge region in an analogous manner to the pyridyl nitrogen (ring B) from imatinib. In addition, the amide connecting the 3-trifluoromethylphenyl group (ring E) and the meta-diaminophenyl (ring D) are expected to form key hydrogen bonds with a glutamate residue in the α-C helix and a backbone amide in the DFG-motif. Finally, the 3-trifluoromethylphenyl group (ring E) is expected to occupy the specificity pocket (Figure 1A).

Table 1

In vitro activities of DSA1-DSA9 and imatinib against Abl, c-Src and Hck. All assays were performed in triplicate or quadruplicate.

In stark contrast to imatinib, DSA1 is a nearly equipotent inhibitor of c-Src (IC₅₀ = 4.6 nM), the Src-family kinase Hck (IC₅₀ = 10 nM) and Abl (IC₅₀ = 10 nM) in an in vitro kinase assay (Table 1). A small panel of triazine derivatives was generated (DSA2-DSA9, Table 1). Variation of the functionality around the pyridinyl triazine core had a small effect on the potency and selectivity of these compounds for Abl, c-Src and Hck, and all of the compounds from this series are equipotent inhibitors of these kinases. Reversing the orientation of the amide that connects the 3-trifluoromethylbenzamide (ring E) and the meta-diaminophenyl ring (ring D) (DSA2) causes a small decrease in potency for all three kinases, but no change in selectivity. Replacing the linkage between these two moieties with a urea group (DSA3), increases the potency for Src, Hck and Abl, but does not alter selectivity. Substituting the meta-diaminophenyl with a meta-aminophenol (DSA6) has little effect on potency or selectivity. Finally, the nature of the functionality that is believed to occupy the specificity pocket seems to have a small effect on the potency and selectivity of these compounds. Clearly, the equipotent inhibition of Abl and c-Src demonstrated by the DSA compounds is not due primarily to compensating binding interactions in the specificity pocket.

Structure of c-Src kinase domain bound to DSA8

The general binding mode of DSA8 in the c-Src•DSA8 complex is similar to that of imatinib in the c-Src•imatinib complex (Figure 4B). DSA8 forms a total of 4 hydrogen bonds with the c-Src kinase domain, and a total of 1294 Å² surface area is buried between the drug and the protein.

Figure 4

Structures of wild type c-Src•imatinib (A, PDB-entry 2OIQ)(3), wild type 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 panel. (more ...)

Rings D- F are chemically identical between imatinib and DSA8 and their interactions with c-Src are essentially the same (Figure 4). Similar to the imatinib complex, the meta-diaminophenyl and benzamide groups (rings D and E) of DSA8 dock into the specificity pocket. The amide carbonyl group that connects rings D and E hydrogen bonds with the backbone of Asp404 in the DFG motif, and the adjacent amino-group hydrogen bonds to the sidechain of Glu310 in helix αC (Figure 4). As for imatinib, the methylpiperazine (ring F) of DSA8 binds to the exposed site.

In DSA8, the triazine and pyridine groups (rings B and C) occupy the adenine binding pocket that imatinib occupies with pyridine and pyrimidine groups (rings B and C) but form different interactions. The pyridine in DSA8 is ortho substituted, whereas the equivalent pyrimidine in imatinib is meta substituted. This leads to a very different accommodation of the pyridine and triazine groups of DSA8 relative to the pyrimidine and pyridine groups in imatinib. The pyridine group of DSA8 fits more deeply into the hydrophobic groove within the adenine pocket, formed by the sidechains of Val281 and Phe405 than the pyrimidine of imatinib.

Inhibitor binding kinetics indicate that DFG-flips are involved in DSA binding

The crystal structures of the complexes of DSA1 and DSA8 demonstrate that the c-Src kinase domain can bind to these inhibitors in the DFG-flipped conformation. One concern arises from the fact that both complexes are in the same crystal form as the c-Src•imatinib complex, raising the possibility that the DFG-flipped conformation is influenced by crystal packing interactions. This is unlikely to be the case, because this crystal lattice is consistent with both active and inactive conformations of c-Src (3). Nevertheless, we sought additional support for the importance of the flipped DFG conformation in the binding of DSA1 and DSA8.

We have developed an assay to monitor the kinetics of inhibitor binding to Abl and Src kinases (3). By choosing conditions under which binding follows pseudo first order kinetics, we can determine the rate constants for binding (k_on) and for dissociation (k_off) (31). For a two state reaction, the dependence of the observed rate constant on inhibitor concentration is linear, and any deviation from linearity indicates additional rate limiting steps. We have shown that for Abl the rate limiting step at high inhibitor concentrations is the DFG-flip rather than the rate of encounter between the inhibitor and the kinase (25). The observed rate constant for imatinib binding to Abl depends nonlinearly on inhibitor concentration, and the binding rate constant decreases with increasing pH. We have shown, using mutagenesis, that this pH dependence is due to deprotonation of the aspartate side chain in the DFG motif, which is expected to thermodynamically disfavor the DFG-Asp out conformation and to effectively slow the DFG flip (25).

We monitored the binding kinetics of DSA1 and DSA8 to the kinase domains of c-Src and Abl (Supplementary Figure S1). We found that the observed rate constants for binding do not increase linearly with inhibitor concentration, consistent with the encounter between the inhibitor and kinase not being rate limiting at high inhibitor concentration. We monitored the binding kinetics at pH 8.0 and pH 5.8, and found that for both c-Src and Abl the binding rate constants decrease with increasing pH, consistent with the DFG-flip being rate limiting, as demonstrated previously for the Abl•imatinib complex (25).

One difference between the binding of imatinib and the DSA compounds to Abl is the 10-fold lower maximal observed rate constant for the DSA compounds (Supplementary Figure S1) (3). This difference may be due to the conformation of the P-loop in the imatinib complex, where it is kinked, and the DSA complexes, where it is extended. We have no experimental information concerning the preferred conformation of the P-loop of Abl in the absence of the inhibitors. One interpretation of our data is that the P-loop of Abl is more stable in the kinked conformation, and that it is in the extended conformation, required for DSA binding, only 10% of the time.

DSA compounds are potent inhibitors of the gatekeeper threonine mutation in c-Src and Abl

The DSA series of inhibitors show in vitro activity against the gatekeeper mutation in both Abl and c-Src kinase domains (Table 2), with IC₅₀ values in the nanomolar range. The gatekeeper residue in c-Src is Thr338. DSA1-DSA9 inhibit c-Src Thr338Ile with only a 2- to 8-fold increase in IC₅₀ value over that for wild type c-Src (Table 2). These compounds also inhibit Abl Thr315Ile with only a 3- to 12-fold lower potency than wild type Abl. Importantly, two of the compounds in this series (DSA3 and DSA7) inhibit full length c-Abl bearing the Thr315Ile mutation with equal or higher potency than that of imatinib for wild type Abl kinase domain in vitro. The kinase domain construct of Abl Thr315Ile could not be expressed in bacteria by our standard method (24), therefore, we used a commercial source of full length Abl Thr315Ile (Invitrogen).

Table 2

Biochemical activities of the DSA compounds against clinically-relevant, imatinib-resistant Abl mutants and the corresponding c-Src mutants.

The crystal structure of DSA1 bound to c-Src Thr338Ile closely resembles that of DSA8 bound to c-Src wt (Figure 4C). A total of 1214 Å² of solvent accessible surface area is buried between protein and DSA1, which lacks the interactions provided by the piperazine group (ring F) in imatinib and DSA8. Instead it is anchored by the trifluoromethyl into a hydrophobic pocket composed of the sidechains of Val313, Leu317, Leu322, Val377, Tyr382, Val402 (Figure 4C).

The reason for the observed gain in activity towards the gatekeeper mutant appears to be due to the rotation of the meta-diaminophenyl (ring D) in DSA1 relative to imatinib (Figure 4). This leads to the displacement of the linker amine group between rings C and D in DSA1 by 1 Å relative to its position in the c-Src•imatinib or Abl•imatinib complexes. While the displacement of the amine linker in DSA1 breaks the hydrogen bond between the sidechain hydroxyl of Thr338 and the linker amine of imatinib, it also prevents the steric clashes that would otherwise occur due to the bulkier isoleucine sidechain in the Thr338Ile gatekeeper mutation. However, the loss of the hydrogen bond to Thr338 is compensated by a hydrogen bond between the amine preceding the trimethoxybenzene (ring A) of DSA1 and the backbone carbonyl of Met341.

The DSA compounds are active against some Abl variants with mutations in the P-loop

Resistance mutations localize most frequently to the P-loop of the Abl kinase domain. We tested the potency of three DSA compounds against Abl kinase domains with the two of the most common mutations in the P-loop (Tyr253His and Glu255Val), which together account for ~30% of the clinically observed resistance mutations (14). The corresponding mutants were also generated in c-Src (Phe278His and Glu280Val). These mutations are likely to disrupt the kinked conformation of the P-loop that is seen in the Abl•imatinib complex (12). The sidechain of Tyr253 stabilizes the P-loop distortion through a hydrogen bond with the backbone and sidechain of Asn322 in the C-lobe and it engages in a perpendicular aromatic-aromatic interaction with the phenylalanine sidechain of the DFG motif. The smaller histidine sidechain cannot engage in either of these interactions and the kinked conformation is likely to be destabilized. The Tyr253His mutant is inhibited in vitro with nanomolar affinity by all the DSA compounds and their potency against this mutant is 10- to 100-fold higher than imatinib (Table 2).

Glu255Val is the most common imatinib-resistant mutation located in the P-loop of Abl. The Glu255 sidechain engages in a hydrogen bonding network with Lys247 and Tyr257 that stabilizes the antiparallel β-strand of the P-loop just before it kinks towards the C-lobe of the kinase domain. The DSA compounds tested show a 2- to 20-fold higher in vitro potency against this mutation than does imatinib (Table 2). Not only are DSA compounds potent against P-loop mutants of Abl, their loss in affinity for these mutants relative to wild type Abl is much less than that of imatinib. We tested the effect of mutations in the c-Src kinase domain on DSA binding (Table 2). Strikingly, the P-loop mutations do not affect the binding of c-Src to the DSA compounds.

The DSA compounds are also active against other resistance mutations, e.g. Phe359Val, which is located in the exposed site (Figure 1A). This mutation was screened against three DSA compounds to test the effect of substitutions on ring E (piperazine vs trifluoromethyl). We find that the DSA compounds are potent inhibitors of the Abl Phe359Val mutation independent of the substitution on ring E (Table 2). Likewise, the DSA compounds tested inhibit c-Src wt and c-Src Tyr382Val with similar potency (Table 2).

DSA compounds inhibit the growth of Ba/F3 cells expressing native and imatinib-resistant BCR-ABL

To determine the cellular efficacy of the DSA compounds, proliferation assays with Ba/F3 cells expressing native BCR-ABL and five imatinib-resistant mutants, including BCR-ABL Thr315Ile, were performed with DSA7 and DSA8 (Figure 4D and Supplementary Figure S2). DSA7 and DSA8 were both found to robustly block the proliferation of all BCR-ABL-expressing cell lines tested. While these compounds were most potent against native BCR-ABL containing cells (IC₅₀ < 10nM), they were also effective at blocking the growth of cells (IC₅₀ < 500nM) harboring Thr315Ile and Glu255Lys mutations in BCR-ABL, which confer a high degree of resistance to imatinib. In stark contrast, the growth of parental Ba/F3 control cells were minimally affected at even the highest concentration tested (5 μM). Consistent with the mechanism of action of these compounds being through the inhibition of BCR-ABL, DSA7 and DSA8 were both found to block the BCR-ABL–induced phosphorylation of CrkL in Ba/F3 cells expressing native and mutant isoforms of BCR-ABL (data not shown). We conclude that DSA compounds effectively inhibit the activity of BCR-ABL and several clinically-relevant mutations in cell-based assays. Importantly, the DSA compounds, like the clinically-approved Abl inhibitors imatinib, dasatinib and nilotinib, do not impair the growth of parental Ba/F3 cells. In contrast, Aurora/Abl inhibitors, the first agents to show preclinical and clinical activity against the BCR-ABL/Thr315Ile mutation, suppress the growth of parental Ba/F3 cells and appear to be toxic to normal hematopoietic elements, thus limiting their utility in patients with CML.

Conclusions

In this study we present a series of inhibitors with two important features. They equipotently inhibit the c-Src and Abl kinase domains in the inactive DFG-Asp out conformation with nanomolar affinity. They are also effective against resistance mutations in Abl, including the Thr315Ile mutation at the gatekeeper position as well as mutations in the P-loop. The sensitivity of the P-loop mutants to the DSA compounds points to the importance of interactions at the adenine pocket, rather than the DFG motif and the specificity site, as being critical for the ability to distinguish between Abl and Src. It appears that the tight binding of Abl requires a high level of solvent exclusion at the adenine pocket. In the Abl•imatinib complex this is provided by the kinked conformation of the P-loop, which effectively closes off a hydrophobic cage for the B and C rings of imatinib (Figure 3) (5). In the c-Kit•imatinib complex the P-loop is in an extended conformation, but an insertion in the activation loop (absent in Abl and c-Src) serves to close off the adenine pocket when imatinib is bound (9). The DSA compounds, which engage the adenine pocket with more polar groups, do not seem to require the formation of this hydrophobic cage.

The key to imatinib selectivity also seems to be its Achilles heel: high affinity imatinib binding to Abl requires solvent shielding through a kinked P-loop conformation which can be disrupted by resistance mutations. This study shows that both c-Src and Abl can readily access the DFG-Asp out conformation and that it is possible to target the clinically important gatekeeper mutation by inhibitors which are specific for the DFG-Asp out conformation. Furthermore, the DSA compounds may prove to be valuable leads for the development of new therapeutics for the treatment of drug-resistant CML as demonstrated by their ability to potently and selectively block the proliferation of Ba/F3 cells expressing clinically-relevant BCR-ABL mutants while sparing parental Ba/F3 cells. Notably, the DSA compounds are able to block the growth of cells expressing the Thr315Ile mutation, which poses an increasingly significant clinical problem.

Supplementary Material

supporting inf

Click here to view.^{(719K, pdf)}

Acknowledgments

Grant Support: NIH R01 grant GM-086858 and the University of Washington (D. J. Maly). NIH 5K99GM080097 (M. A. Seeliger). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

References

1. Sawyers CL, Hochhaus A, Feldman E, et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood. 2002;99:3530–39. [PubMed]

2. Capdeville R, Buchdunger E, Zimmermann J, Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov. 2002;1:493–502. [PubMed]

3. Seeliger MA, Nagar B, Frank F, et al. c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. Structure. 2007;15:299–311. [PubMed]

4. Nagar B, Bornmann WG, Pellicena P, et al. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571) Cancer Res. 2002;62:4236–43. [PubMed]

5. Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science. 2000;289:1938–42. [PubMed]

6. Sicheri F, Moarefi I, Kuriyan J. Crystal structure of the Src family tyrosine kinase Hck. Nature. 1997;385:602–9. [PubMed]

7. Xu W, Harrison SC, Eck MJ. Three-dimensional structure of the tyrosine kinase c-Src. Nature. 1997;385:595–602. [PubMed]

8. De Bondt HL, Rosenblatt J, Jancarik J, et al. Crystal structure of cyclin-dependent kinase 2. Nature. 1993;363:595–602. [PubMed]

9. Mol CD, Dougan DR, Schneider TR, et al. Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase. J Biol Chem. 2004;279:31655–63. [PubMed]

10. Levinson NM, Kuchment O, Shen K, et al. A Src-like inactive conformation in the abl tyrosine kinase domain. PLoS Biol. 2006;4:e144. [PMC free article] [PubMed]

11. Jacobs MD, Caron PR, Hare BJ. Classifying protein kinase structures guides use of ligand-selectivity profiles to predict inactive conformations: structure of lck/imatinib complex. Proteins. 2008;70:1451–60. [PubMed]

12. Shah NP, Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell. 2002;2:117–25. [PubMed]

13. Hochhaus A, La Rosee P. Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance. Leukemia. 2004;18:1321–31. [PubMed]

14. Soverini S, Colarossi S, Gnani A, et al. Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: by the GIMEMA Working Party on Chronic Myeloid Leukemia. Clin Cancer Res. 2006;12:7374–79. [PubMed]

15. Hochhaus A, Kreil S, Corbin AS, et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia. 2002;16:2190–96. [PubMed]

16. Azam M, Seeliger MA, Gray NS, Kuriyan J, Daley GQ. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol. 2008;15:1109–18. [PMC free article] [PubMed]

17. Kantarjian H, Giles F, Wunderle L, et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive. ALL N Engl J Med. 2006;354:2542–51. [PubMed]

18. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science. 2004;305:399–401. [PubMed]

19. Modugno M, Casale E, Soncini C, et al. Crystal structure of the T315I Abl mutant in complex with the aurora kinases inhibitor PHA-739358. Cancer Res. 2007;67:7987–90. [PubMed]

20. Tamborini E, Bonadiman L, Greco A, et al. A new mutation in the KIT ATP pocket causes acquired resistance to imatinib in a gastrointestinal stromal tumor patient. Gastroenterology. 2004;127:294–9. [PubMed]

21. Cools J, DeAngelo DJ, Gotlib J, et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med. 2003;348:1201–14. [PubMed]

22. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7:169–81. [PubMed]

23. Yun CH, Mengwasser KE, Toms AV, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008;105:2070–75. [PubMed]

24. Seeliger MA, Young M, Henderson MN, et al. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 2005;14:3135–9. [PubMed]

25. Shan Y, Seeliger MA, Eastwood MP, et al. A conserved protonation-dependent switch controls drug binding in the Abl kinase. Proc Natl Acad Sci U S A. 2009;106:139–44. [PubMed]

26. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ. Likelihood-enhanced fast translation functions. Acta Crystallographica Section D-Biological Crystallography. 2005;61:458–64. [PubMed]

27. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography. 2004;60:2126–32. [PubMed]

28. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D-Biological Crystallography. 1997;53:240–55. [PubMed]

29. Burgess MR, Skaggs BJ, Shah NP, Lee FY, Sawyers CL. Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance. Proc Natl Acad Sci U S A. 2005;102:3395–400. [PubMed]

30. Hodous BL, Geuns-Meyer SD, Hughes PE, et al. Evolution of a highly selective and potent 2-(pyridin-2-yl)-1,3,5-triazine Tie-2 kinase inhibitor. J Med Chem. 2007;50:611–26. [PubMed]

31. Fersht AR. Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. xxi. New York: W.H. Freeman; 1999. p. 631.

32. Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, Kuriyan J. Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell. 1999;3:639–48. [PubMed]

33. Nagar B, Hantschel O, Young MA, et al. Structural basis for the autoinhibition of cAbl tyrosine kinase. Cell. 2003;112:859–71. [PubMed]

34. Wallace AC, Laskowski RA, Thornton JM. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 1995;8:127–34. [PubMed]