The epidermal growth factor receptor (EGFR) tyrosine kinase has an essential function for the survival of human breast cancer cells. In a systematic effort to design potent and specific inhibitors of this receptor family protein tyrosine kinase (PTK) as antibreast cancer agents, we recently reported the construction of a three-dimensional homology model of the EGFR kinase domain. In this model, the catalytic site is defined by two β-sheets that form an interface at the cleft between the NH2-terminal and COOH-terminal lobes of the kinase domain. Our modeling studies revealed a distinct, remarkably planar triangular binding pocket within the kinase domain with approximate dimensions of 15 Å × 12 Å × 12 Å, and the thickness of the binding pocket is ∼7 Å with an estimated volume of ∼600 Å3 available for inhibitor binding. Molecular docking studies had identified α-cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethoxy)phenyl]-propenamide (LFM-A12) as our lead inhibitor, with an estimated binding constant of 13 μm, which subsequently inhibited EGFR kinase in vitro with an IC50 value of 1.7 μm. LFM-A12 was also discovered to be a highly specific inhibitor of the EGFR. Even at very high concentrations ranging from 175–350 μm, this inhibitor did not affect the enzymatic activity of other PTKs, including the Janus kinases JAK1 and JAK3, the Src family kinase HCK, the Tec family member Bruton’s tyrosine kinase, SYK kinase, and the receptor family PTK insulin receptor kinase. This observation is in contrast to the activity of a quinazoline inhibitor tested as a control, 4-(3-bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline, which was shown to inhibit EGFR and other tyrosine kinases such as HCK, JAK3, and SYK.

EGF2 is a 53-amino acid, single-chain polypeptide (Mr 6,216,000) that exerts biological effects by binding to a specific cell membrane EGFR/ErbB-1 (1, 2, 3, 4). EGFR is a 170-kDa protein, composed of a cysteine-rich, glycosylated extracellular ligand binding domain, a short transmembrane domain, and an intracellular domain, which has tyrosine kinase activity (5). Binding of EGF to the EGFR/ErbB-1 results in receptor dimerization with itself or other members of the Erb-B (subtype I) transmembrane PTK family (e.g., Erb-B2, Erb-B3), resulting in activation and autophosphorylation of the PTK domain (6, 7). Many types of cancer cells display enhanced EGFR expression on their cell surface membranes (3). Enhanced expression of the EGFR on cancer cells has been associated with excessive proliferation and metastasis (4). Examples include breast cancer, prostate cancer, lung cancer, head and neck cancer, bladder cancer, melanoma, and brain tumors (3). In breast cancer, expression of the EGFR is a significant and independent indicator for recurrence and poor relapse-free survival (8, 9, 10). More recently, we and others have shown that the EGFR has an essential function for the survival of human breast cancer (11, 12). Therefore, the development of PTK inhibitors that abrogate the enzymatic function of the EGFR tyrosine kinase has become a focal point in drug discovery research programs aimed at designing more effective treatment strategies for metastatic breast cancer (2, 3, 13, 14).

In a systematic effort to design potent inhibitors of EGFR as antibreast cancer agents, we constructed a three-dimensional homology model of the EGFR kinase domain (Fig. 1,A) and reported (15) a potent inhibitor of the EGFR tyrosine kinase, LFM-A12, targeting the catalytic site of the EGFR kinase (Fig. 1 B). Here, we report that LFM-A12 is a highly specific inhibitor of the EGFR that does not affect the enzymatic activity of other PTKs, including the receptor family PTK IR kinase, the Src family kinase HCK, SYK kinase, the Janus kinases JAK1 and JAK3, and the Tec family kinase BTK, even at concentrations as high as 350 μm. This novel LFM analogue inhibited the proliferation of EGFR-positive human breast cancer cells at micromolar concentrations (15). This observation is in contrast to the activity of a quinazoline inhibitor tested as a control [4-(3- bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline], which was shown to inhibit EGFR as well as other tyrosine kinases such as HCK, JAK3, and SYK. The identification of LFM analogues as EGFR inhibitors with antibreast cancer activity may provide the basis for more effective cancer treatment modalities for breast cancer patients with metastatic disease.

Chemical Synthesis of LFM-A12 and WHI-P154.

The synthesis and characterization of LFM-A12 has been published (15), and WHI-P154 was synthesized and characterized using procedures published previously (16). The chemical structures of LFM-A12 and WHI-P154 are shown in Table 1.

Molecular Modeling Studies.

The homology model for the EGFR kinase domain (Fig. 1 A) was constructed as described previously (15) based on a structural alignment of the sequence of EGFR (accession #P00533, SWISS-PROT; University of Geneva, Geneva, Switzerland) obtained from GenBank (National Center for Biotechnology Information, Bethesda, MD) with the sequences of known crystal structures of other protein kinases [kinase domains of HCK (17), FGFR (18), IR (19), and cAPK (20)]. The modeling was carried out on a Silicon Graphics INDIGO2 computer (Silicon Graphics Inc., Mountain View, CA) using the homology module in INSIGHTII (InsightII Molecular Simulations, Inc. San Diego, CA). The procedure was also used to construct homology models for JAK1, JAK3 (21), BTK (22), and SYK3 [SYK protein sequence was obtained from Rowley et al.(23)].

Fixed docking in the Affinity program within INSIGHTII (InsightII Molecular Simulations, Inc.) was used for docking the compounds LFM-A12 and WHI-P154 to the EGFR tyrosine kinase catalytic site (Fig. 1 B), as described previously (15). The modeling calculations used to study the predicted binding of inhibitors to EGFR were based on the homology model of EGFR and the crystal structure coordinates of LFM-A12 and WHI-P154. LFM-A12 and WHI-P154 were interactively docked into the catalytic site of EGFR based on the position of quercetin in the HCK/quercetin crystal structure (17). As the modeling calculations progressed, the protein residues within a defined radius from the inhibitor were allowed to shift and/or rotate to energetically favorable positions to accommodate the inhibitor. Calculations approximating hydrophobic and hydrophilic interactions were used to determine the 10 best docking positions of each inhibitor in the EGFR catalytic site. The various docked positions of each inhibitor were qualitatively evaluated using a score function in the Ludi module (24, 25) of the program INSIGHTII (InsightII Molecular Simulations, Inc.), which was then used to estimate a binding constant (Ki) for each compound. The above procedure was repeated to dock the inhibitors into the catalytic sites of IR, HCK, BTK, JAK1, JAK3, and SYK.

Tyrosine Kinase Assays.

Immunoprecipitation and kinase assays of recombinant proteins from insect cells expressing BTK, JAK1, or JAK3 were performed as reported previously (26, 27). Antibodies used for immunoprecipitations from insect cells were: polyclonal rabbit anti-BTK serum (28), polyclonal rabbit anti-JAK1 (HR-785, catalogue #sc-277), 0.1 mg/ml rabbit polyclonal IgG affinity purified (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-JAK3 (C-21, catalogue #sc-513), and 0.2 mg/ml rabbit polyclonal IgG affinity purified(Santa Cruz Biotechnology).

EGFR-positive human breast cancer cell line MDA-MB-231 (ATCC HTB-26) was maintained in RPMI 1640 supplemented with 10% fetal bovine serum, as reported earlier (12). For IR kinase assays, HepG2 human hepatoma cells grown to ∼80% confluency were washed once with serum-free DMEM and starved for 3 h at 37°C in a CO2 incubator. Subsequently, cells were stimulated with insulin (Eli Lilly and Co., Indianapolis, IN; catalogue #CP-410;10 units/ml/10 × 106 cells) for 10 min at room temperature. Following this IR kinase activation step, cells were washed once with serum-free medium, lysed in NP40 buffer, and IR was immunoprecipitated from the lysates with an anti-IRB antibody (Santa Cruz Biotechnology; catalogue #sc-711, polyclonal IgG) and assayed for kinase activity as reported (22).

For HCK kinase assays, HCK-transfected COS-7 cells were used as described (22). The cloning and expression of HCK in COS-7 cells has been described previously (29). The pSV7c-HCK plasmid was transfected into 2 × 106 COS-7 cells using lipofectamine (Life Technologies, Inc.), and the cells were harvested 48 h later. The cells were lysed in NP40 buffer, and HCK was immunoprecipitated from the whole cell lysates with an anti-HCK antibody (22, 29).

For EGFR immune complex kinase assays, MDA-MB-231 (breast cancer) cells were stimulated with 10 ng/ml EGF before immunoprecipitation of the EGFR with an anti-EGFR antibody reactive with the sequence Ala351-Asp364 of the human EGFR (Upstate Biotechnology Inc., Lake Placid, NY; catalogue #05–104; Ref. 12). EGFR immune complexes were incubated for 1 h at room temperature with a kinase inhibitor, and tyrosine kinase assays were performed in the presence of [γ-32P]-ATP, as previously described (12, 16, 30, 31). The kinase assay gels were analyzed both by autoradiography and using the Bio-Rad Storage Phosphor Imager System (Bio-Rad, Hercules, CA) for quantitative scanning. The IC50 values were determined using an Inplot program (Graphpad Software, Inc., San Diego, CA).

Modeling Studies of LFM-A12 with the EGFR Kinase Domain.

Our earlier study (15) reported the construction of a novel homology model of the EGFR kinase domain. In this model, the EGFR kinase domain (Fig. 1,A) is composed of a smaller NH2-terminal lobe, which is rich in β-strands, connected by a flexible hinge to a COOH-terminal lobe, which is mostly helical. The catalytic site is defined by two β-sheets that form an interface at the cleft between the two lobes. The catalytic site of the EGFR kinase domain displays a remarkably planar triangular binding pocket, which can bind the base ring portion of ATP. The sides of this triangle are ∼15 Å × 12 Å × 12 Å, and the thickness of the binding pocket is ∼7 Å, with an estimated volume of ∼600 Å3. Two sides of the triangle can be visualized as beginning at an apex located between Thr766 (peach residue in Fig. 1,B) and Asp831 (lavender residue in Fig. 1,B) and extending toward the solvent-accessible opening of the catalytic site. One side of the triangle extends from the apex along the hinge region of the catalytic site (blue residues in Fig. 1,B), and a second side extends from the apex to Arg817 (green residue in Fig. 1 B), which is immediately adjacent to the binding subsites for the sugar and triphosphate groups of ATP. The third side of the triangle extends along the slot-shaped opening to the catalytic site. It is in this catalytic region where small molecule inhibitors can bind.

Our earlier studies (15) had also proposed a binding mode for the leflunomide metabolite analogues on the basis of the constructed three dimensional homology model of the tyrosine kinase domain of EGFR (Fig. 1 A) and had identified the synthetic leflunomide metabolite analogue LFM-A12 as a potent inhibitor of the EGFR tyrosine kinase. The binding mode of LFM-A12 is such that the compound can maintain close contact with the hinge region on the edge of the inhibitor and is sandwiched between the residues Leu694 and Val702 from above and Leu820 and Thr830 from below, and the para-substituted OCF3 group is clamped between the residues Thr766 and Asp831. The nitrile nitrogen of LFM-A12 faces the hinge region and is involved in hydrogen bonding with the amide NH of Met769. In addition, the para-substituted OCF3 group on LFM-A12 can form close contacts between residues Thr766 and Asp831.

Tyrosine Kinase Assays.

We examined the effects of LFM-A12 on the enzymatic activity of the EGFR kinase in cell-free immune complex kinase assays. As shown in Fig. 2,A, a 1-h incubation with LFM-A12 inhibited the EGFR tyrosine kinase in a concentration-dependent fashion in anti-EGFR immunoprecipitates from lysates of MDA-MB-231 human breast cancer cells. The IC50 values for EGFR inhibition were 1.7 μm for LFM-A12 and 5.4 μm for the synthetic unmodified leflunomide metabolite, LFM (Fig. 2,A). We also examined the effects of LFM-A12 on the enzymatic activity of the EGFR tyrosine kinase in breast cancer cells. After a 24-h exposure to LFM-A12, MDA-MB-231 cells were stimulated with EGF for 10 min, and EGFR immune complexes from whole cell lysates were subjected to Western blot analysis with a polyclonal antiphosphotyrosine antibody to measure the autophosphorylation of the EGFR. Treatment of MDA-MB-231 cells with LFM-A12 resulted in markedly decreased tyrosine phosphorylation of the EGFR after EGF stimulation (data not shown). LFM-A12 did not affect the enzymatic activity of other protein tyrosine kinases, including receptor family tyrosine kinase IR (Fig. 2,B), Src family tyrosine kinase HCK (Fig. 2,C), Janus kinases JAK1 and JAK3 (Fig. 2,D–E), Tec family tyrosine kinase BTK (Fig. 2,F), and SYK at concentrations as high as 350 μm(Table 1). In contrast to LFM-A12, the dimethoxyquinazoline compound WHI-P154 inhibited not only EGFR (IC50 = 5.6 μm) but also HCK (IC50 = 11.6 μm), JAK3 (IC50 = 128 μm), and SYK (IC50 = 150 μm; Table 1).

Structural Basis of Specific Inhibition of EGFR Tyrosine Kinase Activity by LFM-A12.

LFM-A12 was found to be a selective inhibitor of EGFR, whereas it was a poor inhibitor of BTK, HCK, JAK1, JAK3, SYK, and IR. We next performed modeling studies using the crystal structure coordinates of HCK (17) and IR (19) and constructed homology models for the kinase domains of JAK1, JAK3 (21), BTK (22), and SYK3 to identify possible causes for the observed selectivity of LFM-A12 for the EGFR tyrosine kinase. LFM-A12 was docked into the kinase domains of EGFR, IR, HCK, JAK1, JAK3, SYK, and BTK. The models were then used to study the binding of LFM-A12 into the catalytic sites of these kinases, and to better understand how LFM-A12 can inhibit EGFR but not BTK, IR, JAK1, JAK3, SYK, or HCK. After energy minimization, the compound maintained favorable close contacts with the hinge region of each kinase. The orientation of LFM-A12 in the catalytic site of BTK and HCK is shown in Fig. 3,A. This orientation as shown in Fig. 3,A is such that the para-substituted OCF3 group of LFM-A12 is below position B and the nitrile group is toward Asp at position C. The peach dotted line in Fig. 3,A represents the hydrogen bond between the BTK residue Thr474 and the hydroxyl group of LFM-A12. There is no hydrogen bond between LFM-A12 and the HCK residues. The orientation of LFM-A12 in the catalytic site of EGFR, IR, JAK1, JAK3, and SYK is shown in Fig. 3,B. This orientation is such that the para-substituted OCF3 group of LFM-A12 is clamped between the residues at positions A and C and the nitrile group is toward the residues at position B. The white dotted line represents the hydrogen bond between the EGFR residue Met769 and the cyano nitrogen of LFM-A12. The green dotted line represents the hydrogen bond between the IR residue Asp1083 and the hydroxyl group of LFM-A12. The cyan dotted line represents the hydrogen bond between the JAK1 residue Glu92 and the hydroxyl group of LFM-A12. There is no hydrogen bonding between LFM-A12 and the residues of SYK and JAK3. Table 1 shows the interaction scores, estimated Ki values, and measured IC50 data for LFM-A12 for the different kinases. Our studies indicated that the selectivity of LFM-A12 for EGFR likely results from its molecular shape and from favorable interactions with unique EGFR residues that are not present in the kinase domains of the other PTKs. Likewise, unfavorable interactions with unique residues of the other PTKs that are not found in the EGFR kinase domain also contribute to this selectivity.

Modeling Studies of WHI-P154 with the EGFR Kinase Domain.

We also studied the kinase inhibition properties of a reference compound belonging to a different class of inhibitors known as 4-anilinoquinazolines (32, 33). The binding of this compound (WHI-P154) with EGFR, JAK1, JAK3, SYK, IR, BTK, and HCK was modeled in a similar way to that of LFM-A12. The experimentally determined IC50 values listed in Table 1 indicated that this compound was capable of inhibiting several kinases and did not demonstrate significant specificity for any one of them. Using the previously described homology models of EGFR, JAK1, JAK3, BTK, and SYK and the crystal structure coordinates of HCK and IR, WHI-P154 was docked into the ATP binding site of these seven kinases. LFM-A12 and WHI-P154 were predicted to bind to the same region of the catalytic site of EGFR, and a large portion of each inhibitor was aligned along the hinge region of the receptor. This position (shown in Fig. 3,C for WHI-P154) favors good contact with the residues in the active site. For WHI-P154 the quinazoline moiety was aligned along the hinge region, and the anilino ring was located between the residues at positions A and C on each side for all of the seven kinases studied. The 6, 7-dimethoxy groups faced the solvent accessible region, and the 4-hydroxyl group was involved in hydrogen bonding with the conserved Asp residue at position C in all seven kinases listed in Table 1. The docked position of WHI-P154 with EGFR revealed a second hydrogen bond between the N3 nitrogen of the quinazoline group and the backbone carbonyl of Met769. For the other six PTKs (HCK, JAK1, JAK3, IR, BTK, and SYK) modeled, these hydrogen bonds do not exist. This maybe due to both the shift of the inhibitor and the displacement of the corresponding residue in SYK, JAK1, JAK3, BTK, IR, and HCK corresponding to Met769 in EGFR due to a bulkier or longer neighboring residue at positions A and/or B. Table 1 shows the interaction scores and estimated Ki values from the modeling studies of WHI-P154 with the seven different kinases. Fig. 3,C shows the superimposed backbones of the ATP binding site residues of the 7 kinases with the docked position of WHI-P154 (multicolor) in EGFR (white). [WHI-P154 was predicted to bind to the catalytic site of HCK (blue), SYK (pink), JAK1 (cyan), JAK3 (red), BTK (peach), and IR (green) in an orientation similar to the EGFR result.). The dotted surface area in Fig. 3 C represents the Connolly surface of WHI-P154. The white dashed lines represent the hydrogen bonds between the EGFR residues and WHI-P154. Our studies have, thus, identified several features that may contribute to the observed inhibition of several kinases by WHI-P154.

Our earlier studies (15) had proposed a binding mode for the LFM analogues on the basis of the three dimensional homology model of the tyrosine kinase domain of EGFR (Fig. 1 A) and had identified the synthetic LFM analogue LFM-A12 as a potent inhibitor of the EGFR tyrosine kinase for which we estimated a Ki value of 13 μm from modeling studies.

Cell-free immune complex kinase assays on the enzymatic activity of the EGFR kinase indicated that 1 h incubation with LFM-A12 inhibited the EGFR tyrosine kinase in a concentration-dependent fashion in anti-EGFR immunoprecipitates from lysates of MDA-MB-231 human breast cancer cells. The IC50 values for EGFR inhibition were 1.7 μm for LFM-A12 and 5.4 μm for the synthetic unmodified leflunomide metabolite LFM (Fig. 2,A). The effects of LFM-A12 on the enzymatic activity of the EGFR tyrosine kinase in MDA-MB-231 breast cancer cells resulted in markedly decreased tyrosine phosphorylation of the EGFR after EGF stimulation (data not shown). The inhibitory effects of LFM-A12 on the EGFR tyrosine kinase were specific in that it did not affect the enzymatic activity of other protein tyrosine kinases, including receptor family tyrosine kinase IR (Fig. 2,B), Src family tyrosine kinase HCK (Fig. 2,C), Janus kinases JAK1 and JAK3 (Fig. 2, D and E), Tec family tyrosine kinase BTK (Fig. 2,F), and SYK at concentrations as high as 350 μm(Table 1).

Modeling studies were performed to understand how LFM-A12 could selectively inhibit EGFR tyrosine kinase, whereas it is a poor inhibitor of BTK, HCK, JAK1, JAK3, SYK, and IR. Whereas most of the catalytic site residues of the EGFR kinase domain were conserved relative to other PTKs, we noted a few specific variations. EGFR residues Leu694, Val702, Lys721, and Ala719 are conserved in EGFR, HCK, FGFR and IR. Residues Asn818 and Asp831 (opposite to the hinge) are conserved in EGFR, HCK, FGFR, IR, BTK, JAK1, JAK3, and SYK. Residues Cys751 and Thr830 are specific for EGFR but vary in BTK (Val, Ser), JAK1 (Val, Gly), JAK3 (Val, Ala), IR (Val, Gly), and HCK (Val, Ala). Residues Thr766 and Leu768 in the hinge region changes to Met and Leu in IR, Met and Phe in JAK1, Met and Tyr in JAK3, to Thr and Tyr in BTK, to Thr and Phe in HCK and to Met and Met in SYK. One region of the binding pocket contains Cys773 in EGFR and is, therefore, considerably more hydrophobic than the corresponding residue of PDGFR (Asp), FGFR (Asn), JAK1 (Ser), HCK (Ser), and IR (Asp).

LFM-A12 was modeled into the kinase domains of IR, HCK, JAK3, JAK1, BTK, SYK, and EGFR. The models were then used to study the binding of LFM-A12 into the catalytic sites of these kinases and to better understand how LFM-A12 can inhibit EGFR but not BTK, IR, JAK1, JAK3, SYK, or HCK. Table 1 shows the interaction scores, estimated Ki values, and measured IC50 data for LFM-A12 with the seven different kinases. After energy minimization, the compound maintained favorable close contacts with the hinge region of each kinase, although the orientation of LFM-A12 in the catalytic site was different for BTK and HCK (as shown in Fig. 3,A) than for EGFR, IR, JAK1, JAK3, and SYK (as shown in Fig. 3,B). Our studies indicated that the selectivity of LFM-A12 for EGFR likely results from its molecular shape and from favorable interactions with unique EGFR residues that are not present in the kinase domains of the other PTKs. Likewise, unfavorable interactions with unique residues of the other PTKs that are not found in the EGFR kinase domain also contribute to this selectivity. These residue differences are illustrated in Fig. 3, A and B, at positions A and B. Fig. 3,A shows the backbone of the EGFR catalytic site, the residue differences between EGFR (white) and other kinases and the docked position of LFM-A12 (multicolor) at this site in BTK (peach), which is also similar to the docked position in HCK (blue). Fig. 3,B shows the docked position of LFM-A12 (multicolor) in EGFR (white), which is also similar to the docked position in JAK1 (cyan), JAK3 (red), SYK (pink), and IR (green). We propose that the aromatic residue in BTK (Tyr) and HCK (Phe; shown at position B in Fig. 3,A) is not as favorable for interactions with the p-OCF3 group of LFM-A12. The corresponding residue in the EGFR kinase domain is leucine (shown in white at position B in Fig. 3,A and 3,B), which would not cause such unfavorable interactions with LFM-A12. Also, for HCK there is a loss of hydrogen bonding interaction with LFM-A12. Furthermore, JAK1, JAK3, IR, and SYK (shown in Fig. 3,B) contain a methionine residue (at position A in Fig. 3,B) that protrudes into the active site and could impair the close hydrophobic contact of LFM-A12 with the hinge region of the catalytic site. The longer methionine residue in these four kinases (JAK1, JAK3, IR, and SYK) does not complement the shape of LFM-A12 and may hinder its binding. As shown in Fig. 3,B, the corresponding residue in the EGFR kinase domain is threonine (white); its relatively shorter side chain enables LFM-A12 (multicolor) to have a more favorable hydrophobic contact with the hinge region which may result in tighter binding to the EGFR binding site. For EGFR, the most active compound (LFM-A12) appears to be located between the residues at positions A and C. Consequently, the estimated Ki value for the EGFR (13 μm) was lower than the Ki values for other PTKs, which ranged from 27 μm for BTK to 1710 μm for JAK3 (Table 1).

On the other hand, a quinazoline inhibitor [4-(3-bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline] tested as a control showed inhibition of EGFR tyrosine kinase, as well as other tyrosine kinases such as HCK, JAK3, and SYK. The experimentally determined IC50 values listed in Table 1 indicated that this compound was capable of inhibiting several kinases and did not demonstrate significant specificity for any one of them. Using the previously described homology models of EGFR, JAK1, JAK3, BTK, and SYK and the crystal structures of HCK and IR, WHI-P154 was docked into the ATP binding site of these seven kinases. Fig. 3,C shows the superimposed backbones of the ATP binding site residues of the seven kinases with the docked position of WHI-P154 (multicolor) in EGFR (white). [WHI-P154 was predicted to bind to the catalytic site of HCK (blue), SYK (pink), JAK1 (cyan), JAK3 (red), IR (green), and BTK (peach) in an orientation similar to the EGFR result]. Table 1 shows the interaction scores and estimated Ki values of WHI-P154 with these kinases. Our studies have identified several features that may contribute to the observed inhibition of several kinases by WHI-P154. The first contributing factor may be the complementary shape of the inhibitor with the hinge region of the binding cavity. The second is the favorable hydrophobic contact between the compound and the residues at positions A and B in Fig. 3,C. The third factor involves hydrogen bonding interactions at position C for all kinases. An additional hydrogen bond with Met769 of EGFR can occur in this region. As seen in Fig. 3,C, the inhibitor WHI-P154 maintains a close contact with the hinge region of all seven kinases. In fact, the longer methionine side chain of JAK1, JAK3, IR, and SYK at position A may serve to enhance the complementary fit of the inhibitor and increase its binding with the corresponding kinase. The same residue at position A prohibits close binding of LFM-A12 to IR, JAK1, JAK3, and SYK in this region. The aromatic residues of HCK, BTK, JAK1, and JAK3 at position B also may enhance the hydrophobic interaction with the quinazoline group of WHI-P154. This is reflected by the corresponding higher hydrophobic interaction scores of this compound for all seven kinases, relative to LFM-A12 (Table 1, Lipo Score). In addition WHI-154 forms 1 or 2 hydrogen bonds with the residues of the corresponding kinase and, like the interaction of LFM-A12 with EGFR, the anilino ring of WHI-P154 appears to be located between the residues at positions A and C for all seven kinases, thereby enhancing close contacts with each of them.

Many classes of small molecule inhibitors have been reported in the last several years for the EGFR kinase; of these, the quinazolines and the pyrazolo/pyrrolo/pyridopyrimidines seem to be the most promising in terms of specificity for inhibiting EGFR. The quinazoline derivative CP-358,774 (11) inhibits EGFR with an IC50 of 2 nm and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nm. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases examined, as determined by assays of isolated kinases and whole cells. Despite the reported profound in vitro potency (Ki = 5 pm) and selectivity of the ATP-competitive brominated quinazoline derivative PD153035 (33), the compound failed to show significant in vivo efficacy. Another type of quinazoline inhibitor reported, PD 168393 and PD 160678, selectively targets and irreversibly inactivates EGFR through covalent modification of a cystein (Cys773) residue present in the ATP binding pocket (34). These compounds also interact in an analogous fashion with erbB2 (which has a conserved Cys residue at the same position) but have no activity against IR, PDGFR, FGFR, and PKC. However, these compounds have not been tested against BTK and JAK3, which also have a conserved cysteine residue at the corresponding position. Two pyrazolopyrimidines have been reported that inhibit EGFR specifically with IC50 values below 10 nm(35) and showed high selectivity toward the tested nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC α, CDK1). Earlier studies have reported that the immunosupressive activity of leflunomide is due to its metabolite A77 1726 (LFM), which is rapidly formed in vivo and functions as a pyrimidine synthesis inhibitor (36) and also inhibits the tyrosine kinase activity of EGFR (37). The compound we report here (LFM-A12) inhibits EGFR with an IC50 in the low micomolar range. Our study is the first that describes the development of LFM analogues as a novel class of EGFR inhibitors which were evaluated using structure-based methods and proposes an explanation for the specificity of LFM-A12 for EGFR.

In summary, our approach led to the successful design of a leflunomide metabolite analogue LFM-A12, which showed selectivity for the EGFR tyrosine kinase. Our results suggest that the selectivity of LFM-A12 for EGFR likely results from its molecular shape and from favorable interactions with unique EGFR residues that are not present in the kinase domains of the other PTKs. Likewise, unfavorable interactions with unique residues of the other PTKs that are not found in the EGFR kinase domain also contribute to this selectivity. This observation is in contrast to the observed inhibition of several kinases (EGFR, HCK, JAK3, and SYK) by WHI-P154. The first contributing factor for the nonselectivity of WHI-P154 may be the complementary shape of the inhibitor with the hinge region of the binding cavity of all seven kinases, which in turn leads to favorable hydrophobic contact between the compound and the residues in this cavity. Additionally, hydrogen bonding interactions with all seven kinases may enhance its binding with each of them. However, the binding volume of the EGFR catalytic site is much larger than the volume occupied by our most potent and selective compound LFM-A12. Increasing the size of the ligand by using larger ring systems might increase the contact area between the receptor and ligand and, hence, enhance binding. Interactions of the inhibitor with the nonconserved residues such as Cys751 and Thr830 in the catalytic site of EGFR may also be used for the design of more potent and selective inhibitors of EGFR.

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2

The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PTK, protein tyrosine kinase; LFM-A12, α-cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethoxy)phenyl]-propenamide; IR, insulin receptor; WHI-P154, 4-(3-bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline; FGFR, fibroblast growth factor receptor.

        
3

C. Mao, unpublished data.

Fig. 1.

A, ribbon (Cα backbone) representation of the homology model of the EGFR kinase domain and a space-filling model of the compound LFM-A12, which was docked into the catalytic site. The NH2-terminal lobe shown in blue is primarily composed of β-sheets, and the COOH-terminal lobe shown in gray is mostly helical. The hinge region is shown in peach. Prepared using Molscript and Raster3D programs (38,39,40). B, space-filling representation of the catalytic site residues of the EGFR kinase domain. The Cα backbone of EGFR is represented as a pink ribbon, and the residues comprising the hinge region are shown in blue. One corner of the triangular binding region (catalytic site) is located between the T766 (peach) and D831 (lavender) residues. The second corner bordering the binding region is located at R817 shown in green, and the third corner is near the lower left side of the hinge region. A ball and stick model of the inhibitor LFM-A12 is shown in multicolor and represents a favorable orientation of this molecule in the kinase active site of EGFR. Prepared using InsightII program (InsightII Molecular Simulations, Inc.).

Fig. 1.

A, ribbon (Cα backbone) representation of the homology model of the EGFR kinase domain and a space-filling model of the compound LFM-A12, which was docked into the catalytic site. The NH2-terminal lobe shown in blue is primarily composed of β-sheets, and the COOH-terminal lobe shown in gray is mostly helical. The hinge region is shown in peach. Prepared using Molscript and Raster3D programs (38,39,40). B, space-filling representation of the catalytic site residues of the EGFR kinase domain. The Cα backbone of EGFR is represented as a pink ribbon, and the residues comprising the hinge region are shown in blue. One corner of the triangular binding region (catalytic site) is located between the T766 (peach) and D831 (lavender) residues. The second corner bordering the binding region is located at R817 shown in green, and the third corner is near the lower left side of the hinge region. A ball and stick model of the inhibitor LFM-A12 is shown in multicolor and represents a favorable orientation of this molecule in the kinase active site of EGFR. Prepared using InsightII program (InsightII Molecular Simulations, Inc.).

Close modal
Fig. 2.

Selective inhibition of EGFR tyrosine kinase by LFM-A12. A, EGFR immune complexes from lysates of MDA-MB-231 human breast cancer cells were treated with LFM or LFM-A12 for 1 h and then assayed for tyrosine kinase activity, as described in “Materials and Methods.” B, lack of inhibition of IR immunoprecipitated from HepG2 hepatoma cells in immune complex kinase assays after treatment with LFM or LFM-A12. C, lack of inhibition of HCK immunoprecipitated from transfected COS7 cells in immune complex kinase assays after treatment with LFM or LFM-A12. D–F, lack of inhibition of JAK3, JAK1, and BTK immunoprecipitated from lysates of transfected insect ovary cells.

Fig. 2.

Selective inhibition of EGFR tyrosine kinase by LFM-A12. A, EGFR immune complexes from lysates of MDA-MB-231 human breast cancer cells were treated with LFM or LFM-A12 for 1 h and then assayed for tyrosine kinase activity, as described in “Materials and Methods.” B, lack of inhibition of IR immunoprecipitated from HepG2 hepatoma cells in immune complex kinase assays after treatment with LFM or LFM-A12. C, lack of inhibition of HCK immunoprecipitated from transfected COS7 cells in immune complex kinase assays after treatment with LFM or LFM-A12. D–F, lack of inhibition of JAK3, JAK1, and BTK immunoprecipitated from lysates of transfected insect ovary cells.

Close modal
Fig. 3.

Docking of LFM-A12 and WHI-P154 into catalytic sites of kinases. A, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), BTK (peach), and crystal structure coordinates of HCK (Ref. 19; blue), with selected residues at positions A, B, and C. LFM-A12 is shown in multicolor and represents its docked position in BTK, which is also similar to its docked position in HCK. The white dotted surface area represents the Connolly surface of LFM-A12. The peach dotted line represents the hydrogen bond between the BTK residue Thr474 and the hydroxyl group of LFM-A12. There is no hydrogen bond between LFM-A12 and the HCK residues. B, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), JAK1 (cyan), JAK3 (red), SYK (pink), and crystal structure coordinates of IR (Ref. 19; green), with selected residues at positions A, B, and C. LFM-A12 is shown in multicolor and represents its docked position in EGFR, which is also similar to its docked position in IR, SYK, JAK1, and JAK3. The white dotted surface area represents the Connolly surface of LFM-A12. The white dotted line represents the hydrogen bond between the EGFR residue Met769 and the cyano nitrogen of LFM-A12. The green dotted line represents the hydrogen bond between the IR residue Asp1083 and the hydroxyl group of LFM-A12. The cyan dotted line represents the hydrogen bond between the JAK1 residue Glu92 and the hydroxyl group of LFM-A12. C, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), JAK1 (cyan), JAK3 (red), SYK (pink), BTK (peach), and crystal structure coordinates of HCK (blue) and IR (green), with selected residues at positions A, B, and C. WHI-P154 is shown in multicolor and represents its docked position in EGFR which is also similar to its docked position in BTK, HCK, SYK, JAK1, JAK3, and IR. The white dotted surface area represents the Connolly surface of WHI-P154. The white dotted lines represent the two hydrogen bonds between the EGFR residues and WHI-P154. The conserved Asp residue at position C is involved in hydrogen bonding with the OH group of the inhibitor in all of the seven kinases.

Fig. 3.

Docking of LFM-A12 and WHI-P154 into catalytic sites of kinases. A, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), BTK (peach), and crystal structure coordinates of HCK (Ref. 19; blue), with selected residues at positions A, B, and C. LFM-A12 is shown in multicolor and represents its docked position in BTK, which is also similar to its docked position in HCK. The white dotted surface area represents the Connolly surface of LFM-A12. The peach dotted line represents the hydrogen bond between the BTK residue Thr474 and the hydroxyl group of LFM-A12. There is no hydrogen bond between LFM-A12 and the HCK residues. B, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), JAK1 (cyan), JAK3 (red), SYK (pink), and crystal structure coordinates of IR (Ref. 19; green), with selected residues at positions A, B, and C. LFM-A12 is shown in multicolor and represents its docked position in EGFR, which is also similar to its docked position in IR, SYK, JAK1, and JAK3. The white dotted surface area represents the Connolly surface of LFM-A12. The white dotted line represents the hydrogen bond between the EGFR residue Met769 and the cyano nitrogen of LFM-A12. The green dotted line represents the hydrogen bond between the IR residue Asp1083 and the hydroxyl group of LFM-A12. The cyan dotted line represents the hydrogen bond between the JAK1 residue Glu92 and the hydroxyl group of LFM-A12. C, superimposed backbones of the catalytic site residues of the kinase domain homology models of EGFR (white), JAK1 (cyan), JAK3 (red), SYK (pink), BTK (peach), and crystal structure coordinates of HCK (blue) and IR (green), with selected residues at positions A, B, and C. WHI-P154 is shown in multicolor and represents its docked position in EGFR which is also similar to its docked position in BTK, HCK, SYK, JAK1, JAK3, and IR. The white dotted surface area represents the Connolly surface of WHI-P154. The white dotted lines represent the two hydrogen bonds between the EGFR residues and WHI-P154. The conserved Asp residue at position C is involved in hydrogen bonding with the OH group of the inhibitor in all of the seven kinases.

Close modal
Table 1

Interaction scores, estimated Ki values, and measured IC50 values for the inhibition of protein tyrosine kinases by LFM-A12 and WHI-P154

Interaction scores, estimated Ki values, and measured IC50 values for the inhibition of protein tyrosine kinases by LFM-A12 and WHI-P154
Interaction scores, estimated Ki values, and measured IC50 values for the inhibition of protein tyrosine kinases by LFM-A12 and WHI-P154
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