3.2 VEGFR-2 Structural Characteristics
First isolated by Terman and colleagues in 1991, VEGFR-2 belongs to the 7-Ig/5-Ig
RTK superfamily, being closely related to previously described class III RTKs, e.g.,
platelet-derived growth factor receptors (PDGFRs), colony-stimulating factor-1
receptor (CSF-1R), and c-KIT receptor [109]. The human VEGFR-2 gene encodes
a full-length protein of 1,356 amino acids, comprising an extracellular (EC) domain
with seven immunoglobulin-like (7-Ig) motifs, a short transmembrane (TM) helix,
and the cytoplasmic subunit. VEGFR-2 is originally translated as a 150 kDa protein,
giving rise to the mature 230 kDa form located in the cell surface after a series of
glycosylations [6, 109–113].
Ligand binding induces receptor dimerization and autophosphorylation of specific intracellular tyrosine residues (Fig. 13), culminating in VEGFR-2 activation
and cell signaling through the intracellular tyrosine kinase (TK) domain. Several
intracellular proteins are phosphorylated by this RTK, including phospholipase Cγ
(PLCγ), phosphatidylinositol 3-kinase (PI3K), p38 mitogen-activated protein kinase
(p38 MAPK), and Ras protein. These signaling proteins trigger downstream pathways that result in increased endothelial cell proliferation, migration, and survival
and vascular permeability, culminating in angiogenesis [6, 109–113].
3.3 VEGFR Inhibitors
The assumption that solid tumors are not able to grow beyond 2 mm in diameter and
are not able to spread to distant sites without developing a vascular supply, provided
by angiogenesis, has indicated the relevance of VEGFR inhibitors (VEGFRi) as
therapeutic tools for cancer treatment. In fact, the efficacy of VEGFRi has been
demonstrated in several preclinical models and in human clinical trials. As previously mentioned, therapeutic approaches to block the VEGF/VEGFR signaling
pathway include those designed against the extracellular ligand-binding domains
of VEGFR, such as monoclonal antibodies, and the small-molecule tyrosine kinase
inhibitors (TKIs) [10, 114, 115]. From the perspective of this chapter, only the latter
approach, i.e., VEGFR TKIs, will be discussed.
The resolution of co-crystal structures of small-molecule TKIs with VEGFR-2
has provided clear structural evidence regarding their general binding mode, guiding
the further design of novel VEGFR-2 inhibitors. In general, the majority of known
VEGFR-2 TKIs bind to the kinase domain through three key hydrogen bonds with
specific amino acid residues, i.e., the backbone NH of hinge residue Cys 919; the
backbone NH of Asp 1046 of the conserved DFG motif; and the side chain
carboxylate of Glu 885. Moreover, a lipophilic pocket defined by residues Ile
888, Leu 889, Ile 898, Val 899, Leu 1019, and Ile 1044 is usually involved in
hydrophobic interactions with ligands’ nonpolar subunits. Finally, hydrophilic contacts with a solvent-exposed region can also be observed for those inhibitors bearing
Case Study on Receptor Tyrosine Kinases EGFR, VEGFR, and PDGFR
173
First isolated by Terman and colleagues in 1991, VEGFR-2 belongs to the 7-Ig/5-Ig
RTK superfamily, being closely related to previously described class III RTKs, e.g.,
platelet-derived growth factor receptors (PDGFRs), colony-stimulating factor-1
receptor (CSF-1R), and c-KIT receptor [109]. The human VEGFR-2 gene encodes
a full-length protein of 1,356 amino acids, comprising an extracellular (EC) domain
with seven immunoglobulin-like (7-Ig) motifs, a short transmembrane (TM) helix,
and the cytoplasmic subunit. VEGFR-2 is originally translated as a 150 kDa protein,
giving rise to the mature 230 kDa form located in the cell surface after a series of
glycosylations [6, 109–113].
Ligand binding induces receptor dimerization and autophosphorylation of specific intracellular tyrosine residues (Fig. 13), culminating in VEGFR-2 activation
and cell signaling through the intracellular tyrosine kinase (TK) domain. Several
intracellular proteins are phosphorylated by this RTK, including phospholipase Cγ
(PLCγ), phosphatidylinositol 3-kinase (PI3K), p38 mitogen-activated protein kinase
(p38 MAPK), and Ras protein. These signaling proteins trigger downstream pathways that result in increased endothelial cell proliferation, migration, and survival
and vascular permeability, culminating in angiogenesis [6, 109–113].
3.3 VEGFR Inhibitors
The assumption that solid tumors are not able to grow beyond 2 mm in diameter and
are not able to spread to distant sites without developing a vascular supply, provided
by angiogenesis, has indicated the relevance of VEGFR inhibitors (VEGFRi) as
therapeutic tools for cancer treatment. In fact, the efficacy of VEGFRi has been
demonstrated in several preclinical models and in human clinical trials. As previously mentioned, therapeutic approaches to block the VEGF/VEGFR signaling
pathway include those designed against the extracellular ligand-binding domains
of VEGFR, such as monoclonal antibodies, and the small-molecule tyrosine kinase
inhibitors (TKIs) [10, 114, 115]. From the perspective of this chapter, only the latter
approach, i.e., VEGFR TKIs, will be discussed.
The resolution of co-crystal structures of small-molecule TKIs with VEGFR-2
has provided clear structural evidence regarding their general binding mode, guiding
the further design of novel VEGFR-2 inhibitors. In general, the majority of known
VEGFR-2 TKIs bind to the kinase domain through three key hydrogen bonds with
specific amino acid residues, i.e., the backbone NH of hinge residue Cys 919; the
backbone NH of Asp 1046 of the conserved DFG motif; and the side chain
carboxylate of Glu 885. Moreover, a lipophilic pocket defined by residues Ile
888, Leu 889, Ile 898, Val 899, Leu 1019, and Ile 1044 is usually involved in
hydrophobic interactions with ligands’ nonpolar subunits. Finally, hydrophilic contacts with a solvent-exposed region can also be observed for those inhibitors bearing
Case Study on Receptor Tyrosine Kinases EGFR, VEGFR, and PDGFR
173