SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo (original) (raw)
Research Articles| December 09 2009
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Grant support: Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract DE-AC02-06CH11357.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address for S.G. Buchanan, J. Hendle, P.S. Lee, C.R. Smith, C.M. Tang, J.M. Sauder, K.D. Schwinn, M. Russell, P.A. Sprengeler, D.A. Thompson, L.A. Pelletier, S. Atwell, S.R. Wasserman, S. Emtage, S.K. Burley, and S.H. Reich: Eli Lilly and Company, San Diego, CA. Current address for K.A. Jessen: Intellikine, Inc., La Jolla, CA.
The atomic coordinates and structure factors were deposited at the Protein Data Bank: accession codes 3DKF, 3DKC, and 3DKG.
Requests for reprints: Sean G. Buchanan, Eli Lilly and Company, 10505 Roselle Street, San Diego, CA 92121. Phone: 858-558-4850; Fax: 858-457-5362. E-mail: buchanan_sean@lilly.com
Received: June 02 2009
Revision Received: September 10 2009
Accepted: October 02 2009
Online ISSN: 1538-8514
Print ISSN: 1535-7163
© 2009 American Association for Cancer Research.
2009
Mol Cancer Ther (2009) 8 (12): 3181–3190.
Revision Received:
September 10 2009
Accepted:
October 02 2009
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Citation
Sean G. Buchanan, Jorg Hendle, Patrick S. Lee, Christopher R. Smith, Pierre-Yves Bounaud, Katti A. Jessen, Crystal M. Tang, Nanni H. Huser, Jeremy D. Felce, Karen J. Froning, Marshall C. Peterman, Brandon E. Aubol, Steve F. Gessert, J. Michael Sauder, Kenneth D. Schwinn, Marijane Russell, Isabelle A. Rooney, Jason Adams, Barbara C. Leon, Tuan H. Do, Jeff M. Blaney, Paul A. Sprengeler, Devon A. Thompson, Lydia Smyth, Laura A. Pelletier, Shane Atwell, Kevin Holme, Stephen R. Wasserman, Spencer Emtage, Stephen K. Burley, Siegfried H. Reich; SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo. _Mol Cancer Ther 1 December 2009; 8 (12): 3181–3190. https://doi.org/10.1158/1535-7163.MCT-09-0477
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Abstract
The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181–90]
Introduction
Hepatocyte growth factor (HGF) receptor, c-Met, or MET, is the prototypic member of a family of receptor tyrosine kinases unique to deuterostomes (1). MET orchestrates the complex program of branching morphogenesis, which is critical during embryonic development and tissue repair and for invasive growth in cancer (2–5). Activating point mutations in the MET gene are found in hereditary and sporadic forms of papillary renal carcinoma (6) and gene amplification leads to upregulated MET signaling in other tumors, notably in the context of acquired resistance to epidermal growth factor receptor inhibitors in lung cancer (7). In other malignancies, such as glioblastoma, autocrine activation of MET by HGF has been shown (8), and MET inhibition is effective in mouse models of glioblastoma (9–12). The wealth of evidence linking MET to cancer has motivated efforts, using various strategies, to inhibit MET signaling (5, 9–16).
ATP-binding pockets of protein kinases possess the appropriate size and physicochemical properties to accommodate small-molecule inhibitors; consequently, considerable medicinal chemistry effort has been directed at ATP-competitive inhibition of various human protein kinases. Although these endeavors have resulted in several revolutionary new drugs, the high degree of amino acid sequence identity within the ATP-binding clefts of canonical protein kinases, presumably reflecting conservation of the phosphotransfer mechanism, presents a major obstacle to the discovery of highly selective, ATP-competitive kinase inhibitors (17). Indeed, all currently approved small-molecule protein kinase inhibitor drugs, and most, if not all, cell-permeable inhibitors that have been used to probe kinase function, target multiple enzymes (18, 19). This promiscuity has often prohibited the incontrovertible assignment of function to a particular kinase activity in physiologic and pathologic processes through the use of ATP-competitive inhibitors (19). The catalytic domain of MET exhibits features common to all protein kinases and presents a similar selectivity challenge. Not surprisingly, therefore, where selectivity has been fully evaluated, cell-permeable MET inhibitors have been shown to target multiple kinases (13–16). As a consequence, where MET kinase function has been implicated in tumorigenesis through the use of such inhibitors, a role of other kinases cannot be ruled out.
Here, we describe SGX523, a novel, ATP-competitive inhibitor that is exquisitely selective for MET. SGX523 is therefore ideally suited to understand the role of MET catalytic activity in tumorigenesis without the confounding effects of off-target kinase inhibition. We use SGX523 to show that the growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers can be inhibited by blocking the catalytic activity of this single receptor tyrosine kinase. Our data also show that truly selective, ATP-competitive MET inhibitors can be found and provide a structural rationale for the selectivity.
Materials and Methods
Protein Expression and Purification
Human MET kinase domain (MET-KD; amino acids 1,067-1,378; NP_001120972) and human RON kinase domain (amino acids 1,053-1,370; NP_002483) were cloned into a custom TOPO-adapted pFastBac vector (Invitrogen) with a NH2-terminal His6 tag and TEV protease cleavage site. The mutations Tyr1212Phe, Tyr1252Phe, and Tyr1253Asp were introduced to make MET-KDmut (20) and a Tyr1248Leu mutation was introduced to make MET-KDTyr1248Leu. Recombinant baculovirus was generated with the Bac-to-Bac system (Invitrogen), and after 48 h expression in Sf9 cells, cell pellets were resuspended in 50 mmol/L Tris-HCl (pH 7.7) and 250 mmol/L NaCl with Complete, EDTA-free protease inhibitor cocktail (Roche). After stirring and centrifugation, the supernatant was bound to Ni-NTA agarose (Qiagen), washed with buffer A [50 mmol/L Tris-HCl (pH 7.8), 500 mmol/L NaCl, 10% glycerol, 10 mmol/L imidazole, and 10 mmol/L methionine], and eluted using a step gradient (buffer A containing 50, 200, and 500 mmol/L imidazole, sequentially). The His6 tag was cleaved overnight at 4°C while dialyzing in buffer A. The resulting sample was passed over a 5 mL IMAC nickel column (Pharmacia) and eluted with an imidazole step gradient followed by gel filtration [HiLoad 16/60 Superdex 200 prep grade equilibrated in 50 mmol/L Tris-HCl (pH 8.5), 150 mmol/L NaCl, 10% glycerol, and 5 mmol/L DTT; Amersham Biosciences]. Protein fractions were combined and concentrated to 10 to 15 mg/mL (Amicon Ultra-15 10,000 Da MWCO). Phosphorylation state was determined by mass spectrometry. Enzyme phosphorylated at three positions, MET-KD(3P), was generated by incubation of MET-KD (1 mg/mL) in 50 mmol/L Tris-HCl (pH 8.5), 150 mmol/L NaCl, 10% glycerol, 5 mmol/L DTT, 2 mmol/L ATP, and 5 mmol/L MgCl2 at room temperature for 15 min followed by addition of EDTA to 0.1 mol/L and removal of ATP, MgCl2, and EDTA with a desalting column.
Kinase Assays
Initial rate constants were measured at 21°C in the presence of 100 mmol/L HEPES (pH 7.5), 0.3 mg/mL poly(Glu-Tyr) peptide substrate, 10 mmol/L MgCl2, 1 mg/mL bovine serum albumin, 5% DMSO, and 20 nmol/L MET-KD and various concentrations of ATP and SGX523. Total reaction volumes (20 μL) were quenched with 20 μL Kinase-Glo detection buffer following the manufacturer's protocol (Promega). Luminescence was detected in a plate-reading luminometer and the results were analyzed by nonlinear regression.
Crystallization and Structure Determination
MET-KDmut with ATP/MgCl2 (4 and 8 mmol/L final concentration) was crystallized by vapor diffusion (with microseeding) against a reservoir containing 11% isopropanol, 2.5% PEG MME 5K, and 100 mmol/L Bis-Tris (pH 6.2). MET-KD, MET-KDmut, and MET-KDTyr1248Leu with SGX523 (1 mmol/L) were crystallized with 20% isopropanol, 200 mmol/L ammonium acetate, and 100 mmol/L Tris-HCl (pH 7.5); 25% (v/v) glycerol was used as a cryoprotectant. Diffraction data were collected at the Advanced Photon Light Source (SGX-CAT 31-ID) and processed using Mosflm. Structures were determined and refined using Xfit and ccp4 (21).
Cell Lines
MDCK canine kidney epithelial cells, MDA-MB-453 breast carcinoma, NCI-H441 human lung carcinoma, U87MG human glioblastoma, and A549 human lung carcinoma cell lines were obtained from the American Type Culture Collection. GTL16 human gastric cancer cells were originally supplied by Dr. Giovanni Gaudino (Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale) and obtained from a collaboration with UroGene. BaF3 cells expressing TPR-MET and TPR-METTyr1248Cys were generated as described by Sattler et al. (22).
Immunoblot Assay
GTL16 cells were grown in RPMI (Invitrogen) supplemented with 10% fetal bovine serum. A549, U87MG, and H441 cells were grown in DMEM (Invitrogen) and 10% fetal bovine serum. Cells (1 × 106) were incubated with various concentrations of SGX523 for 1 h and then treated with lysis buffer (Millipore) plus protease inhibitors (Sigma-Aldrich). Where indicated, 90 ng/mL HGF (USB Corporation) was added for the last 10 min incubation or 150 ng/mL macrophage-stimulating protein (R&D Systems) was added for the last 30 min incubation. Proteins were separated via SDS-PAGE on 4% to 20% gradient gels (80 μg/lane) and standard semidry blotting techniques were used to transfer the protein to nitrocellulose membranes. Phosphorylated signaling molecules were detected using antibodies raised to phospho-MET (Tyr1234/1235), phospho–extracellular signal-regulated kinase (Thr202/Tyr204), phospho-AKT (Ser473), phospho-STAT3 (Tyr705), or phospho-Gab1 (Tyr627) followed by a horseradish peroxidase–conjugated secondary antibody (primary and secondary antibodies were obtained from Cell Signaling). The intensity of the phosphorylated bands was quantified (Typhoon imager and software; GE Life Sciences) and IC50 values were determined by sigmoidal dose-response nonlinear regression analysis (GraphPad Prism 5 Software).
Scatter and Migration Assays
To measure the effect of SGX523 on HGF-induced cell scatter, MDCK cells were plated at 1 × 103 per well in a 24-well plate and incubated at 37°C in 5% CO2 for 1 week in MEM and 10% fetal bovine serum. HGF (90 ng/mL) and various concentrations of SGX523 were added, and the cells were incubated for 18 h (37°C, 5% CO2 humidified incubator) and visualized. To investigate cell migration, A549 cells were plated in 12-well plates (6 × 104 per well) and incubated to confluence. A channel was introduced into the monolayers by scratching with a pipette tip. Various dilutions of compound were added in starve medium in the presence and absence of HGF (90 ng/mL). Twenty-four hours later, wells were checked for cell migration. Cells were stained and visualized as described (13).
In vivo Studies
Female Harlan nude mice (athymic nu/nu; Harlan Laboratories) were s.c. implanted with GTL16, U87, or H441 cells. When tumors had reached ∼150 mm3, SGX523 was administered at various dose regimens by oral gavage. Intratumoral inhibition of MET signaling was determined by measuring average levels of phospho-MET in GTL16 tumors at various time points after the last dose by Western blot analysis. In vivo experiments were conducted at Explora BioLabs in accordance with the requirements for the procurement, housing, care, and use of animals as set forth in the NIH Guide for the Care and Use of Laboratory Animals in Research (revised 1996), the U.S. Department of Agriculture, and the Animal Welfare Act (Public Law: 89-544, 91-579, 91-279 9 CFR parts 1-3).
Results
SGX523 Is an ATP-Competitive Inhibitor of MET
The structure of SGX523 (6-[6-(1-methyl-1_H_-pyrazol-4-yl)-[1,2,4]triazolo-[4,3-_b_]pyridazin-3-ylsulfanyl]quinoline) is shown in Fig. 1A. Enzymatic studies showed that SGX523 potently inhibits the purified MET catalytic domain but not the closely related receptor tyrosine kinase RON (Table 1). SGX523 showed ATP-competitive inhibition (Fig. 1C) with higher apparent affinity for the less active, unphosphorylated form of MET [MET-KD(0P), _K_i = 2.7 nmol/L] versus the more active phospho-enzyme [MET-KD(3P), _K_i = 23 nmol/L; Table 1], a phenomenon consistent with preferential binding to an inactive enzyme conformation.
Figure 1.
![Figure 1. SGX523 is an ATP-competitive inhibitor of MET kinase activity. A, chemical structure of SGX523. B, double reciprocal plots of initial rate constants, kobs, versus ATP (2-500 μmol/L) at varied concentrations of SGX523 [0 nmol/L (Δ), 50 nmol/L (⋄), 100 nmol/L (□), and 200 nmol/L (○)].](3181fig01g.jpeg)
SGX523 is an ATP-competitive inhibitor of MET kinase activity. A, chemical structure of SGX523. B, double reciprocal plots of initial rate constants, _k_obs, versus ATP (2-500 μmol/L) at varied concentrations of SGX523 [0 nmol/L (Δ), 50 nmol/L (⋄), 100 nmol/L (□), and 200 nmol/L (○)].
Figure 1.
SGX523 is an ATP-competitive inhibitor of MET kinase activity. A, chemical structure of SGX523. B, double reciprocal plots of initial rate constants, _k_obs, versus ATP (2-500 μmol/L) at varied concentrations of SGX523 [0 nmol/L (Δ), 50 nmol/L (⋄), 100 nmol/L (□), and 200 nmol/L (○)].
Table 1.
K i values for inhibition of various forms of MET and RON kinase domains by SGX523
| Kinase | SGX523 K_i_ (μmol/L) |
|---|---|
| MET-KD (0P) | 0.0027 |
| MET-KD (3P) | 0.023 |
| MET-KDTyr1248Leu | <10 |
| RON | <10 |
| Kinase | SGX523 K_i_ (μmol/L) |
|---|---|
| MET-KD (0P) | 0.0027 |
| MET-KD (3P) | 0.023 |
| MET-KDTyr1248Leu | <10 |
| RON | <10 |
NOTE: MET-KD is purified from baculovirus as an unphosphorylated enzyme, MET-KD(0P), which is more sensitive to inhibition than the autoactivated form phosphorylated at three sites, MET-KD(3P), as described in Materials and Methods.
SGX523 Is Exquisitely Selective for MET
To determine the kinase selectivity profile of SGX523, the compound was assayed against 213 protein kinases, including wild-type and mutant (Met1268Thr) MET. In this assay, SGX523 inhibits wild-type MET with an IC50 of 4 nmol/L. At a screening concentration of 1,000 nmol/L, no protein kinase from the panel, other than MET or METMet1268Thr, was inhibited by >36%, implying that the IC50 values for all other enzymes in the panel exceed ∼1 μmol/L (Supplementary Fig. S1). Formal IC50 determinations were conducted for the five most sensitive kinases (Supplementary Table S1). No significant enzyme inhibition was observed, even at compound concentrations up to ≥7 μmol/L. Therefore, SGX523 exhibits >3 orders of magnitude selectivity for MET versus approximately half of all catalytically competent human protein kinases and three quarters of all human protein tyrosine kinases.
To quantify and compare the selectivity profiles of different kinase inhibitors, Karaman et al. (18) developed a “Selectivity Score” parameter. Previously, lapatinib was the most selective kinase inhibitor described (score = 0.01). For SGX523, the Karaman score = 0.005, making it by far the most selective small-molecule inhibitor of MET and, in fact, more selective than all other ATP-competitive kinase inhibitors for which selectivity profiles across comparably large, diverse enzyme panels have been published.
SGX523 Stabilizes a Unique, Inactive Conformation of the MET Catalytic Domain
X-ray crystallographic study of SGX523 bound to the catalytic domain of MET at 1.8 Å resolution verified that the inhibitor does indeed bind to the ATP site of an inactive conformation (Fig. 2A-D). The two bicyclic ring systems of SGX523 define two distinct, orthogonal binding elements. The quinoline moiety engages the MET hinge region with a single canonical hydrogen bond between the quinoline nitrogen and backbone carbonyl of Met1178 and a nonclassic C-H-O=C hydrogen bond between quinoline C8 and the carbonyl oxygen of Pro1176 (Fig. 2D). Thus, interactions made by the quinoline moiety closely resemble interactions formed by other ATP-competitive kinase inhibitors at the “hinge region” (23) and do not explain the observed selectivity. The triazolopyridazine ring system, on the other hand, is pinioned between the hydrophobic side chains of Met1229 and Tyr1248 stabilizing a highly unusual activation loop trajectory and an enzyme conformation not observed in any protein kinase structure in the Protein Data Bank besides MET. Further stabilizing this binding mode, the triazolopyridazine moiety of the inhibitor forms a classic hydrogen bond to Asp1240 and two nonclassic C-H-O=C hydrogen bonds can be identified in a bifurcated interaction where the triazolopyridazine C-H and a pyrazole C-H both traject toward the carbonyl oxygen of Arg1226 (Fig. 2C).
Figure 2.
Binding mode of SGX523. A, ribbon diagram of SGX523 bound to the ATP-binding region of MET kinase (Protein Data Bank ID: 3DKF). α-helix C (17) is labeled with an arrow. The backbone of the activation loop is colored magenta. B, two-dimensional representation of SGX523 in the MET binding pocket. All side chains within 4.5 Å of the inhibitor are shown in an image generated by the Molecular Operating Environment, Chemical Computing Group (Quebec, Montreal, Canada, 2007). C, detailed view of the binding site as shown in A to depict the key interactions of the triazolopyridazine ring system with Tyr1248 of the activation loop (magenta backbone). D, binding site is rotated to depict the interactions of the kinase hinge region with the quinoline ring including the nonclassic C-H-O=C hydrogen bond (see text).
Figure 2.
Binding mode of SGX523. A, ribbon diagram of SGX523 bound to the ATP-binding region of MET kinase (Protein Data Bank ID: 3DKF). α-helix C (17) is labeled with an arrow. The backbone of the activation loop is colored magenta. B, two-dimensional representation of SGX523 in the MET binding pocket. All side chains within 4.5 Å of the inhibitor are shown in an image generated by the Molecular Operating Environment, Chemical Computing Group (Quebec, Montreal, Canada, 2007). C, detailed view of the binding site as shown in A to depict the key interactions of the triazolopyridazine ring system with Tyr1248 of the activation loop (magenta backbone). D, binding site is rotated to depict the interactions of the kinase hinge region with the quinoline ring including the nonclassic C-H-O=C hydrogen bond (see text).
SGX523 is unusual in that, despite showing higher affinity for unphosphorylated enzyme, it stabilizes a “DFG-in” conformation normally observed for inhibitors that bind an active conformation and do not discriminate between phosphorylated and unphosphorylated kinases (23). Nevertheless, the activation loop conformation stabilized by SGX523 is quite distinct from that seen in the active conformation of other protein kinases. Because there is no published structure of MET in its active conformation, we determined the structure of the MET:Mg2+:ATP ternary complex to compare the active conformation to the SGX523-bound form (Supplementary Fig. S2A). Tyr1248 of the activation loop moves >14 Å on binding SGX523 to occupy the location of the triphosphate moiety of ATP in the ternary complex, a conformation clearly incompatible with phosphotransfer and thus “inactive.”
Tyr1248 Is Essential for the High-Affinity Binding of SGX523 to MET
The π-π interaction between Tyr1248 and the triazolopyridazine ring system of the inhibitor likely plays a critical role in stabilizing the unusual activation loop contortion. RON, the nearest neighbor of MET in the kinome (24), possesses a Leu in the position equivalent to Tyr1248 and is not significantly inhibited by SGX523 at concentrations up to 10 μmol/L (Table 1). To further investigate the importance of Tyr1248, a Tyr1248Leu mutant form of MET was expressed, purified, and tested for inhibition by SGX523. SGX523 was also tested in cells expressing an oncogenic MET fusion protein, TPR-MET, bearing an analogous Tyr1248Cys substitution. Tyr1248 mutations rendered MET insensitive to inhibition by SGX523 in purified enzyme (Table 1) and cell-based assays (IC50 > 10 μmol/L; see below). Determination of an X-ray crystal structure of SGX523, at millimolar concentration, bound to the Tyr1248Leu form of MET revealed that, while hinge binding of SGX523 is preserved, the activation loop adopts a completely disordered conformation with no detectable electron density for Leu1248. These data show that Tyr1248 is essential for the high-affinity interaction of SGX523 with the MET-KD.
No Other Human Protein Kinase Shares the MET Active Site Features Required for SGX523 Binding
To further understand the origin of the selectivity of SGX523, we performed a bioinformatic analysis of all human protein kinases. First, we identified all residues within 4.5 Å of SGX523 (Fig. 2B). Three of these 16 contact residues, Arg1226 and Asn1227, from the catalytic loop, and Asp1240, the first residue of the “DFG” signature motif, are highly conserved among protein tyrosine kinases and are therefore unlikely to account for the selectivity of SGX523. Of the remaining 13 contact residues, the energetic importance of Tyr1248 was described above and we reasoned that any other protein kinase susceptible to SGX523 inhibition would possess an aromatic residue at the equivalent position. Forty-nine other human protein kinases possess either tyrosine (27 examples) or another aromatic residue (Phe, His, or Trp; 22 examples). We examined these 49 enzyme sequences for additional similarity among the other 12 SGX523 contact residues. No other protein kinase shares precisely the same constellation of 13 side chains comprising the MET active site (Table 2). RON differs only at one position, with a leucine at the side chain equivalent to Tyr1248, and is insensitive to inhibition as described above. AXL, MER, and TYRO3 share 9 of the 13 side chains and are the next most closely related enzymes (24). We computed homology models for each of these AXL family members using our structure of the SGX523-bound form of MET as a modeling template. In all three cases, and in most of the 27 protein tyrosine kinases listed in Table 2, the side chain equivalent to Ala1244 of MET is bulkier than alanine. The additional space occupied by the serine hydroxyl of AXL, MER, and TYRO3 versus Ala1244 appears incompatible with SGX523 binding. Similar analyses of all 46 other protein kinases sharing an aromatic residue analogous to Tyr1248 revealed likely steric incompatibility with the mode of SGX523 binding observed for MET. With the sole exception of MET, we have documented that >200 protein kinases are not sensitive to SGX523 inhibition. Our bioinformatics analysis suggests that the remaining members of the human protein kinome are similarly insensitive to SGX523 inhibition.
Table 2.
List of all human kinases sharing a tyrosine side chain at the position equivalent to MET Tyr1248 and annotated for conservation of the side chains lining the SGX523:MET binding pocket
| Kinase | No. identities* | I1102 | V1110 | A1126 | L1158 | L1175 | P1176 | Y1177 | M1178 | D1182 | M1229 | A1239 | A1244 | Y1248 | % Inhibition at 1 μmol/L SGX523 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MET | 13 | - | - | - | - | - | - | - | - | - | - | - | - | - | 92 |
| RON | 12 | - | - | - | - | - | - | - | - | - | - | - | - | L | 9 |
| AXL | 9 | L | - | - | M | - | - | F | - | - | - | - | S | - | ND† |
| MER | 9 | L | - | - | I | - | - | F | - | - | - | - | S | - | 7 |
| TYRO3 | 9 | L | - | - | A | - | - | F | - | - | - | - | S | - | 19 |
| MUSK | 8 | - | - | - | V | F | E | - | - | - | L | - | S | - | -4 |
| ROS | 8 | L | - | - | - | - | E | L | - | - | L | G | - | - | 7 |
| ALK | 7 | L | - | - | V | - | E | L | - | - | L | G | - | - | -10 |
| LTK | 7 | L | - | - | V | - | E | L | - | - | L | G | - | - | ND |
| DDR1 | 7 | L | - | - | I | T | D | - | - | - | L | - | S | - | ND |
| DDR2 | 7 | L | - | - | I | T | E | - | - | - | L | - | S | - | ND |
| IGF1R | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | -3 |
| INSR | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | -1 |
| IRR | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | 5 |
| TRKA | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 10 |
| TRKB | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 0 |
| TRKC | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 7 |
| LMR1 | 6 | - | - | V | - | M | E | F | C | - | L | G | - | - | ND |
| LMR3 | 6 | - | - | V | - | M | E | F | C | - | L | G | - | - | ND |
| MOK | 6 | - | - | - | - | C | E | L | - | N | L | G | C | - | ND |
| FLT1 | 5 | L | - | - | V | V | E | - | C | N | L | C | - | - | 0 |
| ROR2 | 5 | L | - | - | V | F | S | - | C | - | L | S | F | - | ND |
| PIK3R4 | 5 | L | - | V | - | R | Q | - | V | N | - | T | F | - | ND |
| CCK4 | 5 | L | - | L | V | - | E | - | V | - | L | S | S | - | ND |
| SuRTK106 | 5 | - | I | I | V | - | E | D | V | - | L | C | - | - | ND |
| FLT4 | 4 | L | - | - | V | V | E | F | C | N | L | C | - | - | 1 |
| RET | 4 | L | - | - | I | V | E | - | A | S | L | S | S | - | 5 |
| ROR1 | 4 | L | I | - | V | F | E | - | I | - | L | S | S | - | ND |
| KDR | 4 | L | - | - | V | V | E | F | C | N | L | C | - | - | 5 |
| Kinase | No. identities* | I1102 | V1110 | A1126 | L1158 | L1175 | P1176 | Y1177 | M1178 | D1182 | M1229 | A1239 | A1244 | Y1248 | % Inhibition at 1 μmol/L SGX523 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MET | 13 | - | - | - | - | - | - | - | - | - | - | - | - | - | 92 |
| RON | 12 | - | - | - | - | - | - | - | - | - | - | - | - | L | 9 |
| AXL | 9 | L | - | - | M | - | - | F | - | - | - | - | S | - | ND† |
| MER | 9 | L | - | - | I | - | - | F | - | - | - | - | S | - | 7 |
| TYRO3 | 9 | L | - | - | A | - | - | F | - | - | - | - | S | - | 19 |
| MUSK | 8 | - | - | - | V | F | E | - | - | - | L | - | S | - | -4 |
| ROS | 8 | L | - | - | - | - | E | L | - | - | L | G | - | - | 7 |
| ALK | 7 | L | - | - | V | - | E | L | - | - | L | G | - | - | -10 |
| LTK | 7 | L | - | - | V | - | E | L | - | - | L | G | - | - | ND |
| DDR1 | 7 | L | - | - | I | T | D | - | - | - | L | - | S | - | ND |
| DDR2 | 7 | L | - | - | I | T | E | - | - | - | L | - | S | - | ND |
| IGF1R | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | -3 |
| INSR | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | -1 |
| IRR | 6 | L | - | - | V | M | E | L | - | - | - | G | T | - | 5 |
| TRKA | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 10 |
| TRKB | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 0 |
| TRKC | 6 | L | - | - | V | F | E | - | - | - | L | G | S | - | 7 |
| LMR1 | 6 | - | - | V | - | M | E | F | C | - | L | G | - | - | ND |
| LMR3 | 6 | - | - | V | - | M | E | F | C | - | L | G | - | - | ND |
| MOK | 6 | - | - | - | - | C | E | L | - | N | L | G | C | - | ND |
| FLT1 | 5 | L | - | - | V | V | E | - | C | N | L | C | - | - | 0 |
| ROR2 | 5 | L | - | - | V | F | S | - | C | - | L | S | F | - | ND |
| PIK3R4 | 5 | L | - | V | - | R | Q | - | V | N | - | T | F | - | ND |
| CCK4 | 5 | L | - | L | V | - | E | - | V | - | L | S | S | - | ND |
| SuRTK106 | 5 | - | I | I | V | - | E | D | V | - | L | C | - | - | ND |
| FLT4 | 4 | L | - | - | V | V | E | F | C | N | L | C | - | - | 1 |
| RET | 4 | L | - | - | I | V | E | - | A | S | L | S | S | - | 5 |
| ROR1 | 4 | L | I | - | V | F | E | - | I | - | L | S | S | - | ND |
| KDR | 4 | L | - | - | V | V | E | F | C | N | L | C | - | - | 5 |
*The list of all human protein kinases collated by Manning et al. (24) was scanned to locate amino acids at the positions equivalent to the 13 side chains forming the MET:SGX523 binding site with the Pfam Pkinase Hidden Markov model. The 27 kinases with a tyrosine at the position equivalent to the MET activation loop Tyr1248 were ranked according to number of identities at positions equivalent to the remaining 12 side chains forming the MET:SGX523 pocket. RON is the only kinase sharing twelve of the thirteen amino acids seen in MET but differs at Tyr1248.
†ND, not determined.
We also compared the shape of the SGX523 binding pocket in MET to the shapes of binding pockets identified in other structurally characterized protein kinase/inhibitor complexes. We examined all protein kinase structures found in the Protein Data Bank that exhibit inactive conformations (25) and compared them to the MET:SGX523 structure. We found that the MET:SGX523 binding pocket shape is distinct from all other non-MET protein kinase cocrystal structures in the Protein Data Bank, including structures of other inhibitors that stabilize DFG-in conformations, such as erlotinib (Supplementary Fig. S2B). Thus, to accommodate SGX523, MET uniquely combines a DFG-in conformation with a highly unusual activation loop contortion.
SGX523 Inhibits MET, but not Other Kinases, in Cells
The exquisite selectivity of SGX523 suggests that it should inhibit MET-dependent cellular functions without affecting other signal transduction pathways. To first show that SGX523 could directly inhibit MET catalytic activity in cells, BaF3 murine pro-B cells were transfected with TPR-MET, which encodes a constitutively active fusion protein (22, 26, 27), and with a version of TPR-MET substituted at the position equivalent to Tyr1248 to mimic the naturally occurring oncogenic form TPR-METTyr1248Cys (28–30), which should not bind SGX523. BaF3 cells expressing either form of TPR-MET showed high levels of autophosphorylation. SGX523 potently reduced the autophosphorylation signal in cells expressing the normal form but had no effect, at concentrations up to 10 μmol/L, in cells expressing the Tyr1248Cys form (Supplementary Fig. S3). These results indicate that SGX523 binds and inhibits the MET-KD in cells.
SGX523 was next tested in cell lines derived from human cancers. MET, activated by HGF or by overexpression, stimulates signaling via the MAPK, AKT, and other pathways (2, 5). In the gastric cancer cell line GTL16, MET gene amplification leads to constitutive signaling, which is abolished by SGX523 (Fig. 3A). The IC50 value for the inhibition of MET autophosphorylation was 0.040 μmol/L in GTL16 cells (Supplementary Fig. S4). SGX523 also abrogates HGF-induced signaling in A549 lung cancer cells (Fig. 3B), with an IC50 value of 0.012 μmol/L for the inhibition of MET autophosphorylation. An indirect reduction of levels of total protein cannot explain this decrease. As shown in Supplementary Fig. S5, brief (1 h) exposure to SGX523 inhibits MET autophosphorylation without affecting total MET or extracellular signal-regulated kinase protein levels. Longer exposure to SGX523 is associated with increased levels of total MET presumably due to relief from negative feedback regulation (data not shown). At 1 μmol/L, SGX523 had no effect on signaling events stimulated by macrophage-stimulating protein, the RON ligand (Fig. 3C), nor signaling by eight other protein kinases (data not shown).
Figure 3.
SGX523 inhibits MET signaling in cells. A, SGX523 inhibition of constitutive MET signaling in human gastric cancer cells. GTL16 cells (1 × 106) were incubated with various concentrations of SGX523 for 1 h and MET autophosphorylation and downstream signaling were analyzed by immunoblot. The intensity of the phospho-MET band was quantified and an IC50 value for inhibition of MET autophosphorylation was determined (IC50 = 0.040 μmol/L). B, SGX523 inhibition of MET signaling in A549 lung carcinoma cells. A549 cells (1 × 106) were incubated with various concentrations of SGX523 for 1 h with or without 90 ng/mL HGF. MET autophosphorylation and downstream signaling were analyzed by immunoblot (phospho-MET IC50 = 0.012 μmol/L). C, SGX523 does not inhibit signaling stimulated by the RON ligand macrophage-stimulating protein. MDA-MB-453 cells were serum-starved overnight before being treated with SGX523 for 1 h and stimulated with macrophage-stimulating protein (150 ng/mL) followed by immunoblot analysis to detect activation of RON signaling.
Figure 3.
SGX523 inhibits MET signaling in cells. A, SGX523 inhibition of constitutive MET signaling in human gastric cancer cells. GTL16 cells (1 × 106) were incubated with various concentrations of SGX523 for 1 h and MET autophosphorylation and downstream signaling were analyzed by immunoblot. The intensity of the phospho-MET band was quantified and an IC50 value for inhibition of MET autophosphorylation was determined (IC50 = 0.040 μmol/L). B, SGX523 inhibition of MET signaling in A549 lung carcinoma cells. A549 cells (1 × 106) were incubated with various concentrations of SGX523 for 1 h with or without 90 ng/mL HGF. MET autophosphorylation and downstream signaling were analyzed by immunoblot (phospho-MET IC50 = 0.012 μmol/L). C, SGX523 does not inhibit signaling stimulated by the RON ligand macrophage-stimulating protein. MDA-MB-453 cells were serum-starved overnight before being treated with SGX523 for 1 h and stimulated with macrophage-stimulating protein (150 ng/mL) followed by immunoblot analysis to detect activation of RON signaling.
The proliferation of gastric and lung cancer cells with amplification of the MET gene, but not cell lines with normal MET gene copy number, is sensitive to MET-directed RNA interference and multitargeted MET kinase inhibitors (31, 32). SGX523 inhibits the growth of gastric and lung cancer cell lines with amplification of the MET gene but has no effect, even at high micromolar concentration, on cell lines with normal MET gene copy number (Supplementary Table S3). These results suggest that tumors with MET amplification may respond to exquisitely selective MET kinase inhibitor drugs.
SGX523 also potently inhibited HGF-dependent motility. HGF has been shown to dissociate and scatter epithelial cell aggregates (33). SGX523 prevented HGF-induced scattering of MDCK canine kidney epithelial cells (Fig. 4A) and prevented HGF-promoted migration across a gap artificially introduced in a monolayer of A549 lung carcinoma cells (Fig. 4B).
Figure 4.
SGX523 inhibits scattering and migration of cells in response to HGF. A, MDCK cells were grown as small colonies at low density and treated with HGF (90 ng/mL) in the presence of various concentrations of SGX523. After an overnight incubation, cells were visualized. B, confluent monolayers of A549 cells were scratched using a pipette tip to introduce a gap. SGX523 was added at various concentrations in the presence and absence HGF (90 ng/mL). Twenty-four hours later, the monolayer gap was visualized. Magnification, ×40 (A and B). Experiments were done at least three times with very similar results.
Figure 4.
SGX523 inhibits scattering and migration of cells in response to HGF. A, MDCK cells were grown as small colonies at low density and treated with HGF (90 ng/mL) in the presence of various concentrations of SGX523. After an overnight incubation, cells were visualized. B, confluent monolayers of A549 cells were scratched using a pipette tip to introduce a gap. SGX523 was added at various concentrations in the presence and absence HGF (90 ng/mL). Twenty-four hours later, the monolayer gap was visualized. Magnification, ×40 (A and B). Experiments were done at least three times with very similar results.
SGX523 Inhibits MET-Dependent Tumor Growth
Human GTL16 gastric cancer cells, which exhibit MET gene amplification, form tumors in mice. As shown in Fig. 5A, SGX523 significantly retards the growth of preestablished GTL16 tumors when administered orally at doses of ≥10 mg/kg twice daily. Inhibition of MET catalytic activity was assessed in the same experiment by measuring levels of autophosphorylated MET in the GTL16 tumor lysates after 16 days of oral administration of SGX523 at the doses indicated (Fig. 5B). As can be seen in comparing Fig. 5A and B, potent inhibition of MET kinase activity was associated with thorough tumor growth inhibition. Interestingly, treatment of mice using a daily schedule of 60 mg/kg was also highly effective in retarding tumor growth despite incomplete target inhibition through the entire 24 h dosing interval. Xenografts derived from U87MG human glioblastoma cells, which activate MET by an autocrine mechanism (8), and H441 human lung cancer cells, which express phospho-MET (34), are sensitive to multitargeted kinase inhibitors (14, 15, 34). The growth of U87MG tumors is additionally impeded by ribozymes and antibodies directed at HGF (9, 10) or MET (9, 12). These data suggest that U87MG tumors depend on MET activity for their growth, but biologic agents targeting receptor tyrosine kinases can act via mechanisms other than inhibition of catalysis. To determine whether selective inhibition of MET kinase activity was sufficient to prevent the growth of U87MG- and H441-derived xenografts, SGX523 was administered to nude mice bearing established tumors (Fig. 5C and D). At a dose of 10 mg/kg administered twice daily, SGX523 potently inhibited U87MG tumor growth; at 30 mg/kg dosed twice daily, SGX523 led to clear regression of U87MG tumors (Fig. 5C). SGX523, dosed twice daily at 30 mg/kg, also retarded the growth of H441 tumors (Fig. 5D) with concomitant reduction in tumor MET autophosphorylation levels (Supplementary Fig. S6). Together, these results confirm that specific blockade of MET kinase activity can effectively inhibit the growth of tumors in which MET is activated by distinct mechanisms.
Figure 5.
SGX523 inhibits MET-dependent tumor growth. A, growth of GTL16 tumor xenografts in mice treated with SGX523 at doses of 10, 20, 30, and 100 mg/kg twice daily or 60 mg/kg daily by oral gavage starting at day 4. Average ± SE tumor volume (n = 12). P < 0.001. **B,** intratumoral inhibition of MET signaling. After 14 d of SGX523 treatment, tumors (_n_ = 3 per dose per time point) were excised and average levels of phospho-MET (_p-MET_) were determined by immunoblot. For all twice daily dose schedules, the decreased tumor growth was accompanied by thorough (>90%) inhibition of tumor phospho-MET levels. C, growth of U87MG tumor xenografts in mice treated with SGX523 at doses of 10 or 60 mg/kg twice daily by oral gavage starting at day 5 (arrow) for 22 d. Average ± SE tumor volume (n = 5). D, growth of H441 tumor xenografts in mice treated with 60 mg/kg twice daily SGX523 from day 5 (arrow) for 22 d. Average ± SE tumor volume (n = 5).
Figure 5.
SGX523 inhibits MET-dependent tumor growth. A, growth of GTL16 tumor xenografts in mice treated with SGX523 at doses of 10, 20, 30, and 100 mg/kg twice daily or 60 mg/kg daily by oral gavage starting at day 4. Average ± SE tumor volume (n = 12). P < 0.001. **B,** intratumoral inhibition of MET signaling. After 14 d of SGX523 treatment, tumors (_n_ = 3 per dose per time point) were excised and average levels of phospho-MET (_p-MET_) were determined by immunoblot. For all twice daily dose schedules, the decreased tumor growth was accompanied by thorough (>90%) inhibition of tumor phospho-MET levels. C, growth of U87MG tumor xenografts in mice treated with SGX523 at doses of 10 or 60 mg/kg twice daily by oral gavage starting at day 5 (arrow) for 22 d. Average ± SE tumor volume (n = 5). D, growth of H441 tumor xenografts in mice treated with 60 mg/kg twice daily SGX523 from day 5 (arrow) for 22 d. Average ± SE tumor volume (n = 5).
Discussion
We have identified and characterized a novel, potent, ATP-competitive inhibitor of the MET receptor tyrosine kinase. In an extensive survey of >200 human kinases, we have found no other enzyme sensitive to this inhibitor, even at concentrations 3 orders of magnitude over the IC50 for MET. Analysis of crystal structures of SGX523 bound to the MET-KD revealed an unusual conformation that has not been observed in any other protein kinase and suggested that an important contribution to the binding affinity derives from Tyr1248. Mutation of Tyr1248 confirmed the critical contribution of this side chain to SGX523 binding. The few human kinases that have an aromatic side chain at the position equivalent to METTyr1248 differ in other regions of the SGX523 binding pocket, which sterically preclude binding to the compound with high affinity. This analysis not only explains the observed selectivity but also suggests that, aside from MET, no human kinase can be potently inhibited by SGX523. Also, these results, combined with the observations that MET catalytic activity tolerates mutation of Tyr1248 and that mutations affecting this position have been identified in human tumors (28–30), suggest that missense mutation at codon 1,248 in the MET gene is a likely mechanism of resistance to inhibitors of the SGX523 class.
The novel mode of protein kinase binding we have detailed here was recently described for two other small molecules that inhibit MET and appear to rely on similar π-π interactions with Tyr1248 (15, 35). Bicyclic triazoles, chemically related to SGX523, were also described at a recent meeting (36, 37). These new compounds, which have not yet been characterized to the same extent as SGX523, may also be highly selective.
SGX523 is one of the most selective, ATP-competitive kinase inhibitors ever described and the most selective small-molecule inhibitor of MET. Devoid of the confounding effects of promiscuous kinase inhibition, SGX523 is an ideal compound for dissecting the role of MET catalytic activity in normal physiology and disease and for guiding the use of MET kinase inhibitors in the treatment of cancer. SGX523 is orally bioavailable in all species tested and thus serves as a useful tool to probe the role of MET in animal models. We used SGX523 to show that tumors derived from human glioblastoma and lung and gastric cancers depend on MET catalytic activity for their growth. Our results suggest that clinical trials of exquisitely selective MET kinase inhibitors should include cancers showing MET activation by amplification or autocrine signaling. Regrettably, a phase I clinical trial to evaluate the safety of SGX523 had to be discontinued due to kidney toxicity. This unexpected effect is thought to have resulted from accumulation of a metabolite in humans, which was not observed at significant levels during animal toxicology studies. The suspect metabolite, which does not inhibit MET, is highly insoluble and may have crystallized in renal tissue. A detailed report of the clinical studies will be the subject of a separate article.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
David Smith, John Koss, and Sonal Sojitra helped in collecting X-ray diffraction data at the SGX Collaborative Access Team; the beam line was constructed and operated by SGX Pharmaceuticals at Sector 31 of the Advanced Photon Source.
References
1
D'Aniello
S
,
Irimia
M
,
Maeso
I
, et al.
Gene expansion and retention leads to a diverse tyrosine kinase superfamily in amphioxus
.
Mol Biol Evol
2008
;
25
:
1841
–
54
.
2
Birchmeier
C
,
Birchmeier
W
,
Gherardi
E
,
Vande Woude
GF
.
Met, metastasis, motility and more
.
Nat Rev Mol Cell Biol
2003
;
4
:
915
–
25
.
3
Gentile
A
,
Trusolino
L
,
Comoglio
PM
.
The Met tyrosine kinase receptor in development and cancer
.
Cancer Metastasis Rev
2008
;
27
:
85
–
94
.
4
Ma
PC
,
Maulik
G
,
Christensen
J
,
Salgia
R
.
c-Met: structure, functions and potential for therapeutic inhibition
.
Cancer Metastasis Rev
2003
;
22
:
309
–
25
.
5
Comoglio
PM
,
Giordano
S
,
Trusolino
L
.
Drug development of MET inhibitors: targeting oncogene addiction and expedience
.
Nat Rev Drug Discov
2008
;
7
:
504
–
16
.
6
Schmidt
L
,
Duh
FM
,
Chen
F
, et al.
Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas
.
Nat Genet
1997
;
16
:
68
–
73
.
7
Engelman
JA
,
Zejnullahu
K
,
Mitsudomi
T
, et al.
MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling
.
Science (New York)
2007
;
316
:
1039
–
43
.
8
Koochekpour
S
,
Jeffers
M
,
Rulong
S
, et al.
Met and hepatocyte growth factor/scatter factor expression in human gliomas
.
Cancer Res
1997
;
57
:
5391
–
8
.
9
Abounader
R
,
Lal
B
,
Luddy
C
, et al.
In vivo targeting of SF/HGF and c-met expression via U1snRNA/ribozymes inhibits glioma growth and angiogenesis and promotes apoptosis
.
FASEB J
2002
;
16
:
108
–
10
.
10
Burgess
T
,
Coxon
A
,
Meyer
S
, et al.
Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors
.
Cancer Res
2006
;
66
:
1721
–
9
.
11
Cao
B
,
Su
Y
,
Oskarsson
M
, et al.
Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models
.
Proc Natl Acad Sci U S A
2001
;
98
:
7443
–
8
.
12
Martens
T
,
Schmidt
NO
,
Eckerich
C
, et al.
A novel one-armed anti-c-Met antibody inhibits glioblastoma growth in vivo
.
Clin Cancer Res
2006
;
12
:
6144
–
52
.
13
Christensen
JG
,
Schreck
R
,
Burrows
J
, et al.
A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo
.
Cancer Res
2003
;
63
:
7345
–
55
.
14
Zou
HY
,
Li
Q
,
Lee
JH
, et al.
An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms
.
Cancer Res
2007
;
67
:
4408
–
17
.
15
Bellon
SF
,
Kaplan-Lefko
P
,
Yang
Y
, et al.
c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations
.
J Biol Chem
2008
;
283
:
2675
–
83
.
16
LoRusso P, Appleman L, Heath E, et al. A phase I study of a novel spectrum selective kinase inhibitor (SSKI), XL880, administered orally in patients with solid tumors. AACR-NCI-EORTC; 2005 Nov 14-18; Pennsylvania Convention Center, Philadelphia, PA; 2005.
17
Hanks
SK
,
Hunter
T
.
Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification
.
FASEB J
1995
;
9
:
576
–
96
.
18
Karaman
MW
,
Herrgard
S
,
Treiber
DK
, et al.
A quantitative analysis of kinase inhibitor selectivity
.
Nat Biotechnol
2008
;
26
:
127
–
32
.
19
Bain
J
,
Plater
L
,
Elliott
M
, et al.
The selectivity of protein kinase inhibitors: a further update
.
Biochem J
2007
;
408
:
297
–
315
.
20
Schiering
N
,
Knapp
S
,
Marconi
M
, et al.
Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a
.
Proc Natl Acad Sci U S A
2003
;
100
:
12654
–
9
.
21
Collaborative Computational Project Number 4
.
The CCP4 suite: programs for protein crystallography
.
Acta Crystallogr D Biol Crystallogr
1994
;
50
:
760
–
3
.
22
Sattler
M
,
Pride
YB
,
Ma
P
, et al.
A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase
.
Cancer Res
2003
;
63
:
5462
–
9
.
23
Liu
Y
,
Gray
NS
.
Rational design of inhibitors that bind to inactive kinase conformations
.
Nat Chem Biol
2006
;
2
:
358
–
64
.
24
Manning
G
,
Whyte
DB
,
Martinez
R
,
Hunter
T
,
Sudarsanam
S
.
The protein kinase complement of the human genome
.
Science (New York)
2002
;
298
:
1912
–
34
.
25
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
.
26
Park
M
,
Dean
M
,
Cooper
CS
, et al.
Mechanism of met oncogene activation
.
Cell
1986
;
45
:
895
–
904
.
27
Ma
PC
,
Schaefer
E
,
Christensen
JG
,
Salgia
R
.
A selective small molecule c-MET Inhibitor, PHA665752, cooperates with rapamycin
.
Clin Cancer Res
2005
;
11
:
2312
–
9
.
28
Schmidt
L
,
Junker
K
,
Nakaigawa
N
, et al.
Novel mutations of the MET proto-oncogene in papillary renal carcinomas
.
Oncogene
1999
;
18
:
2343
–
50
.
29
Jeffers
M
,
Schmidt
L
,
Nakaigawa
N
, et al.
Activating mutations for the met tyrosine kinase receptor in human cancer
.
Proc Natl Acad Sci U S A
1997
;
94
:
11445
–
50
.
30
Di Renzo
MF
,
Olivero
M
,
Martone
T
, et al.
Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas
.
Oncogene
2000
;
19
:
1547
–
55
.
31
Lutterbach
B
,
Zeng
Q
,
Davis
LJ
, et al.
Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival
.
Cancer Res
2007
;
67
:
2081
–
8
.
32
Smolen
GA
,
Sordella
R
,
Muir
B
, et al.
Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752
.
Proc Natl Acad Sci U S A
2006
;
103
:
2316
–
21
.
33
Stoker
M
,
Gherardi
E
,
Perryman
M
,
Gray
J
.
Scatter factor is a fibroblast-derived modulator of epithelial cell mobility
.
Nature
1987
;
327
:
239
–
42
.
34
Puri
N
,
Khramtsov
A
,
Ahmed
S
, et al.
A selective small molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts
.
Cancer Res
2007
;
67
:
3529
–
34
.
35
Albrecht
BK
,
Harmange
JC
,
Bauer
D
, et al.
Discovery and optimization of triazolopyridazines as potent and selective inhibitors of the c-Met kinase
.
J Med Chem
2008
;
51
:
2879
–
82
.
36
Perera
T
,
Lavrijssen
T
,
Janssens
B
, et al.
JNJ-38877605: a selective Met kinase inhibitor inducing regression of Met-driven tumor models
.
2008
.
37
Zou
HY
,
Li
Q
,
Joseph
L
, et al.
PF-04217903, a novel selective c-Met kinase inhibitor with potent antitumor and anti-angiogenic properties in vitro and in vivo
.
2008
.
Competing Interests
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
© 2009 American Association for Cancer Research.
2009