Structure and clinical relevance of the epidermal growth factor receptor in human cancer

Abstract

Purpose: To review the recent advances in the atomic-level understanding of the epidermal growth factor receptor (EGFR) tyrosine kinase (TK). We aim to highlight the current and future importance of these studies for the understanding and treatment of malignancies where EGFR-TK is improperly activated.

Methods: The analysis was conducted on published crystal structures deposited in the Protein Data Bank (www.pdb.org) using the program O.

Results: In this review we emphasize how recent EGFR kinase domain crystal structures can explain the mechanisms of activation for L858R and other EGFR-TK mutations, and compare these distinct activating mechanisms with those recently described for the wild-type EGFR. We suggest an atomic-level mechanism for the poor efficacy of lapatinib against tumors with activating EGFR kinase domain point mutations compared with the efficacy of gefitinib and erlotinib, and demonstrate how structural insights help our understanding of acquired resistance to these agents. We also highlight how these new molecular-level structural data are expected to affect the development of EGFR-TK targeted small molecule kinase inhibitors.

Conclusion: There are now more crystal structures published for the EGFR-TK domain than for any other TK. This wealth of crystallographic information is beginning to describe the mechanisms by which proper regulation of EGFR-TK is lost in disease. These crystal structures are beginning to show how small molecules inhibit EGFR-TK activity and will aid development of EGFR-TK mutant targeted therapies.

Figures

Figure 1. EGFR tyrosine kinase crystal structures

A. Cartoon illustrating the active state locations of the major structural regions of EGFR-TK discussed in this review. The position of an ATP analogue, AMP-PNP, in the catalytic cleft is shown (ATP) and the locations of the catalytic glutamic acid (Glu) and lysine (Lys) residues are shown (PDB accession code of structure used: 1ITW). B and C. Ribbon representation of the two crystallized conformations of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase. Panel B shows the crystal structure of EGFR tyrosine kinase in complex with erlotinib (PDB accession code: 1M17). Panel C shows the crystal structure of EGFR tyrosine kinase in complex with lapatinib (PDB accession code: 1XKK). The kinase N- and C-lobes and C-helix are indicated, the activation loop is colored in gold and the glycine rich P-loop, blue. EGFR tyrosine kinase inhibitors erlotinib and lapatinib are shown as space-filling spheres. Locations of activating leucine-747 to glutamic acid-749 'LRE' deletion and leucine-858 to arginine (L858R) point mutation are shown in stick format in red and are labeled. The conformation of the two crystal structures differs with the activation loop of the erlotinib-bound structure seen in an active-like conformation and the activation loop of the lapatinib-bound crystal structure trapped in an inactive conformation. All structural figures made using the program PYMOL (http://www.pymol.org). Panel D shows a schematic of EGFR family activation based on crystal structures reviewed in Hubbard, 2005 69 and here. On extracellular ligand binding the receptor dimerizes allowing the cytoplasmic EGFR-TK to activate in a tail-to-head fashion. The locations of regions within EGFR-TK that we discuss are indicated on the exon boundary map.

Figure 2. Activation of CDKs and EGFRs occurs by a similar mechanism

Panel A. Crystal structures of inactive CDK2 (PDB accession code: 1HCK) and cyclin-A in complex with CDK2 (PDB accession code: 1FIN). Panel B. Crystal structures of inactive EGFR tyrosine kinase domain (PDB accession code: 1XKK) and active state EGFR-TK (PDB accession code: 2GS6). There is a striking similarity between the inactive states of EGFR-TK and CDK2. Activation of these kinases is achieved by a protein-protein interaction that forces a structural rearrangement of the kinase towards the active state. In this figure kinase activation loops are colored orange and the C-helix is colored red. The catalytic glutamic acid is shown as space-filling spheres. The important conformational movements of the C-helix and the activation loop are indicated, but other conformational movements that occur between the active and inactive states are not shown. The location of the catalytic cleft is indicated. It is important to note that this activating mechanism for wild-type EGFR is different from the disease-associated EGFR mutations.

Figure 3. Crystal structures suggest mechanisms of activation and reduced inhibitor sensitivity

A and B. In the crystal structure of the EGFR tyrosine kinase domain bound to lapatinib (PDB accession code: 1XKK) leucine-858 is located in a hydrophobic pocket. This pocket is defined by residues phenylalinine-723 (F723), leucine-747 (L747), methionine-766 (M766), leucine-788 (L788), leucine-861 (L861), leucine-862 (L862), a salt bridge between lysine-745 (K745) and aspartate-855 (D855), and the hydrophobic 3-fluorobenzyl-oxy group of EGFR tyrosine kinase-specific inhibitor. Panel A shows the residues which define this pocket in a stick-like format and Panel B depicts them as space-filling spheres. Leucine-858 is shown in stick format in red in both panels and lapatinib is shown as space-filling spheres in both panels. Secondary structure coloring as per Figure 1. Mutation of the small hydrophobic leucine-858 to a large charged arginine residue is expected to destabilize this conformation and push the kinase conformational equilibrium towards the active state. Pane C. The replacement of leucine-858 with an arginine residue is depicted. An arginine residue is shown in purple (with nitrogen atoms in blue) to illustrate that the relative size and charge of this residue are poorly compatible with this inactive conformation. This is panel is a model and not based on experimental diffraction data. Panel D. Difference in position of the C-helix from the erlotinib- and lapatinib-bound crystal structures of EGFR. The C-helices of the erlotinib- and lapatinib-bound structures are colored green and blue respectively. Residue methionine-742 (M742) is expected to sterically clash with lapatinib when the kinase is in the active conformation, with the expected result that lapatinib would bind poorly to the active conformation. The inset shows a close up of this clash. For this panel, crystal structures 1M17 and 1XKK were superimposed using the program TOPP. Panel E. The most commonly seen resistance mutation to ATP-competitive inhibitors in EGFR-TK is threonine-790 to methionine. The location of this amino acid residue is deep in the catalytic cleft, but is predicted to deleteriously affect the binding of small molecule inhibitors gefitinib and erlotinib. Irreversible inhibitors to EGFR-TK have been shown to surmount this resistance and covalently bind to cysteine-797. Here we have illustrated these points by showing the molecular surface of active state EGFR in grey (PDB accession code: 1M17), the location of cysteine-797 as a yellow patch, and the location of the acquired methionine residue as red spheres (the orientation of this residue is modeled based on the crystal structure of insulin receptor kinase, 1IRK, which is in a similar conformation and has a methionine at this location). The blue mesh indicates the location of erlotinib bound to active state EGFR-TK. Covalent inhibitor binding to cysteine-797 will occur proximal to the kinase catalytic cleft.