Receptor tyrosine kinases (RTKs) are part of the larger family of protein tyrosine kinases. They are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Approximately 20 different RTK classes have been identified.[1]
Lapatinib is a EGFR inhibitor used in breast cancer treatment. ERBB2 is necessary for heart cells proliferation and regeneration[2].
Epidermal Growth Factor Receptors are overexpressed in many types of human carcinomas including lung, pancreatic, and breast cancer, and are often mutated. This overexpression leads to excessive activation of the anti-apoptotic Ras signalling cascade, resulting in uncontrolled DNA synthesis and cell proliferation. Studies have revealed that the is responsible for activating this Ras signaling cascade. Upon binding ligands like Epidermal Growth Factor, EGFR dimerizes and autophosphorylates several tyrosine residues at its C-terminal domain. Upon phosphorylation, EGFR undergoes a significant conformational shift, revealing an additional binding site capable of binding and activating downstream signaling proteins.[3][4] Erlotinib inhibits the EGFR tyrosine kinase by located within the kinase domain. Residues Met 774, Leu 825, Val 707, Thr 835, Asp 836, Phe 837, Thr 771, Lys 726, Ala 724, & Leu 769 tightly bind the inhibitor in place. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated.[5][6]
Gefitinib inhibits the EGFR tyrosine kinase by located within the kinase domain. Residues Lys 745, Leu 788, Ala 743, Thr 790, Gln 791, Met 193, Pro 794, Gly 796, Asp 800, Ser 719, Glu 762, & Met 766 tightly bind the inhibitor. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated.[7]
Erlotinib inhibits the EGFR tyrosine kinase by located within the kinase domain. EGFR uses residues Asp 831, Lys 721, Thr 766, Leu 820, Gly 772, Phe 771, Leu 694, Pro 770, Met 769, Leu 768, Gln 767 & Ala 719 to tightly bind the inhibitor. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated.[8][9]
A Possible Strategy against Head and Neck Cancer: In Silico. Investigation of Three-in-One inhibitors[10]
(colored in darkmagenta), which is an enzyme with decarboxylation reaction of uroporphyrinogen III to (colored in salmon), is overexpressed in tumor tissues and has potential to sensitize cancer patients to radiotherapy. Moreover, (colored in magenta) and (colored in deeppink), which are tyrosine kinase receptors in the erbB family, are also overexpressed in tumor tissues and have been indicated as the important targets of therapy for cancer. In this research, we discuss the possible conformation for an inhibitor against three target proteins, UROD, EGFR, and Her2.
Virtual screening of the UROD (PDB ID: 1r3y), EGFR (PDB ID: 3poz), and Her2 (PDB ID: 3pp0) was conducted using the binding site defined by the volume and location of the co-crystallized compounds in each crystal structure. In silico results indicate the traditional Chinese medicine (TCM) compounds had high binding affinity with all three target protein. For (colored in magenta), the top three compounds, (colored in yellow), (colored in cyan), and (colored in orange), formed hydrogen bonds with the residues, Arg803, Lys913 and some other residues in the binding domain. The docking poses of (colored in deeppink) with (colored in yellow), (colored in cyan), and (colored in orange), exhibited hydrogen bonds between ligands and the residues in the binding site. For protein (colored in darkmagenta), (colored in yellow), (colored in cyan), and (colored in orange), have hydrogen bonds with the three important binding and catalytic residues Arg37, Arg41, Tyr164, and the residue His220. The three TCM compounds hint towards a probable molecule backbone which might be used to evolve drug-like compounds against EGFR, Her2, and UROD, and have potential application against head and neck cancer.
See also Herceptin - Mechanism of Action
is a hormone made of two separate amino acid chains that are bound by multiple disulfide bonds. Insulin is synthesized and secreted from the islets of Langerhans of the pancreas in response to high concentrations of glucose in the blood.
The insulin receptor is a dimer of made of two and two . [11] Within the extracellular ectodomain, there are four potential that can interact with insulin ligands on the extracellular side of the membrane. The full extracellular and intracellular components of the insulin receptor have only been imaged in separate sections but a larger picture of how these sections combine to initiate downstream tyrosine autophosphorylation is emerging.[12]
The make up the extracellular domain (ectodomain) of the insulin receptor and are the sites of insulin binding. The alpha subunit is comprised of two Leucine rich domains (L1 & L2), a Cysteine rich domain (CR), and a .[13] α-CT has a unique position that allows it to reach across the receptor and interact with the insulin at the binding site on the opposing side of the receptor. The alpha subunits are held together by a disulfide bond between on each alpha subunit. The disulfide bonds are important to the overall stabilization of the molecule as it binds to insulin. Two types of insulin binding sites are present in the alpha subunits, and (Figure 2). The sites are in pairs because of the heterodimeric nature of the receptor. Due to structural differences, as well as greater surface area and accessibility, binding sites 1 and 1' have much higher affinity for insulin binding than sites 2 and 2'. Insulin can also bind at sites 2 and 2', but the location on the back of the beta sheet of the FnIII-1 domain and lack of surface area decreases the likelihood of their binding site becoming occupied as quickly.[14]
The spans from the extracellular domain across the transmembrane region and into the intracellular portion of the insulin receptor. The beta subunit is composed of part of fibronectin domain III-2 and all of Fibronectin domain III-3.[13] The beta subunit's FnIII-3 domain has links through the transmembrane region into the intracellular part of the membrane. Cryo-EM provided clear representations of the FnIII-2 and FnIII-3 domains (Figure 1) but are missing the transmembrane and intracellular regions. Although the FnIII-3 domain is connected to the transmembrane and intracellular regions, the active conformation likely extends all the way to the tyrosine kinase domain region (see PDB 4XLV).[15]
The alpha and beta subunits of the extracellular domains fold over one another and form a when the insulin receptor is inactivated. Upon activation, the extracellular domain undergoes a conformational change and forms a . An additional component to the ectodomain is .[14] Each of the dimers has an α-CT helix. The α-CT helix is a single alpha-helix that plays an important role in insulin binding and stabilization of the "T" shape activated conformation. α-CT interacts with a leucine-rich region of the alpha subunit and a fibronectin type III region of the beta subunit to form the insulin binding sites known as .[14]
The structure of the extracellular domain is stabilized through multiple disulfide bonds. The alpha subunits are linked through two disulfide bonds, with the main one being between of two adjacent alpha subuntis [16]. of both alpha subunits are also held together with a disulfide bond.[17] The alpha subunit is also attached to the beta subunit by a disulfide bond between the .[17]
The insulin receptor unit has four separate sites for the insulin binding. There are two pairs of two identical binding sites referred to as and .
The insulin molecules bind to these sites mostly through hydrophobic interactions, with some of the most crucial residues at sites 1 and 1' being between of the insulin receptor FnIII-1 domain.[14] Despite some of the residues included being charged, the main interactions are still hydrophobic in this binding site. For example, due to arginine carrying its positive charge at the end of the side chain, to allow the hydrophobic part of the side chain to interact with the other hydrophobic residues. The alpha subunits also have significant that help maintain a compact binging site. At sites 2 and 2', the major residues contributing to these hydrophobic interactions are the .[14]
Sites 1 and 1' have a higher binding affinity than sites 2 and 2' due to site 1 having a larger surface area (706 Å2) exposed for insulin to bind to compared to site 2 (394 Å2).[14] The binding interactions of the insulin molecules in sites 1 and 1' are facilitated by hydrophobic residues of an of the insulin receptor. The insulin molecules in sites 2 and 2' primarily interact with the residues that comprise some of the of the insulin receptor.
At , a occurs between three critical parts of the alpha subunits of the insulin receptor.[14] The entire interface of the tripartite interaction involves many residues that are involved with intra-protomer ionic and hydrogen bonding at the binding site. The α-CT chain and the FnIII-1 domain region come into close proximity during the conformational change of the insulin receptor and their interaction involves the following residues: and the . This duo then interacts with the L1 region, specifically ARG14, creating an ideal for the insulin ligand. The FnIII-1 and α-CT are interacting from the two different alpha subunits, which displays a "cross linking" scenario where the domains of the heterodimer can intertwine with each other. The tripartite interaction between α-CT, the FnIII-1 domain, and the L1 region is important because it allows for a strong interaction between two subunits of the insulin receptor that maintains and stabilizes the T-shape activation state for the rest of the downstream signaling to occur.[14]
It has been hypothesized that activation of the insulin receptor can change based on the concentration of insulin. These recent cryo-EM structures of the insulin receptor have demonstrated that at least three insulin molecules have to bind to the insulin receptor to induce the active conformation, as binding of two insulin molecules is insufficient to induce a full conformational change.[14] However, this conclusion has not yet been widely confirmed.[14] In low concentrations of insulin, the insulin receptor may not require binding of three insulin molecules in order to exhibit activation. Rather, the level of activity will change in accordance to the availability of insulin.[14] When higher concentrations of insulin are present, the conformational difference between the two-insulin-bound state and the three-insulin-bound state is drastic as the insulin receptor transitions from the inactive to the active .[14] However, in conditions of low insulin availability, the two-insulin-bound state may be enough to induce partial activation of the receptor.[14]
The conformational change between the inverted, inactive and the active of the insulin receptor is induced by insulin binding. The T shape conformation is well observed in the alpha subunit. It is horizontally composed of L1, CR (including the ), and L2 domains and vertically composed of the FnIII-1, 2, and 3 domains (Figure 1). The proper conformational change of the ectodomain of the insulin receptor is crucial for transmitting the signal into the cell. The movements extracellularly cause the two receptor tyrosine kinase domains intracellularly to become close enough to each other to autophosphorylate. This autophosphorylation activates the tyrosine kinase domain, initiating intracellular insulin signaling cascades.
When an insulin molecule binds to site 1 of the alpha subunit, the respective protomer is recruited and a slight inward movement of the of the beta subunit is initiated. This is accomplished by the formation of several salt bridges, specifically between .[14] Binding of insulin to both protomers establishes a full activation of the insulin receptor. This activation is demonstrated through the inward movement of both protomers. This motion has been referred to as a "hinge" motion as both protomers "swing" in towards one another.[14] Figure 4 depicts the conformational change and "hinge motion" between the inactive and active forms of an insulin receptor protomer. Upon insulin binding, the beta subunits of the inactive form, shown in blue, are "swung" inward to the active form, shown in orange. When the receptor is in an , the FnIII-3 domains are separated by about 120Å.[18] This distance prevents the initiation of autophosphorylation and downstream signaling by the tyrosine kinase domains on the intracellular side of the receptor. Upon the binding of insulin to multiple binding sites, this conformation change brings the FnIII-3 domains within 40Å of each other to induce the conformation.[14] [19] As the fibronectin type III domains of the beta subunit swing inward, the alpha subunits also undergo a conformational change upon insulin binding. As insulin binds to site 1, the leucine-rich region of one protomer interacts with α-CT and the FNIII-1 domains of the other protomer to form the binding site.[14] For the tripartite interface to form, the alpha subunits of each protomer must undergo a "folding" motion. While snapshots of various conformational states of the insulin receptor have been captured, the complex dynamics of the insulin receptor conformational changes upon insulin binding are still being actively investigated.[14]
(PDB entry 3bu5).
TK domain of IR contains an activation loop and a catalytic loop and . The bound IR substrate 2 peptide tyrosine is the phosphorylated residue[20].
In type 2 diabetes, the TK domain is thought to be down-regulated through phosphorylation of by protein kinase C[21].
. Water molecules shown as red spheres.
Vascular Endothelial Growth Factor Receptors (VEGFRs) are tyrosine kinase receptors responsible for binding with VEGF to initiate signal cascades that stimulate angiogenesis among other effects[22]. VEGFRs convey signals to other signal transduction effectors via autophosphorylation of specific residues in its structure. Because VEGFRs are up-regulated in cancerous tumors which have a high metabolic need for oxygen, VEGFRs are an important target for pharmaceutical drugs treating cancer. VEGFR subtypes are numbered 1,2,3. The VEGFRs are a family of tyrosine kinase receptors on the surface of different cells depending on family identity. VEGFR-1 is expressed on haematopoietic stem cells, monocytes, and vascular endothelial cells. VEGFR-2 is expressed on vascular endothelial cells and lymphatic endothelial cells, while VEGFR-3 is only expressed on lymphatic endothelial cells[23]. The structure of VEGFR-2 can been seen at the right. VEGF-A binds to the second and third extracellular Ig-like domains of VEGFR-2 with a 10-fold lower affinity than it does to the second Ig-like domain of VEGFR-1, despite the fact that VEGFR-2 is the principal mediator of several physiological effects on endothelial cells including proliferation, migration, and survival.[24] Binding of VEGF to the domains 2 and 3 of a VEGFR-2 monomer increases the probability that an additional VEGFR-2 binds the tethered ligand to form a dimmer. Once the two receptors are cross-linked, interactions between their membrane-proximal domain 7s stabilize the dimmer significantly. This dimerization and stabilization allows for precise positioning of the intracellular kinase domains, resulting in autophosphorylation and subsequent activation of the classical extracellular signal-regulated kinases (ERK) pathway.[25].
The tyrosine kinase domain of VEGFR-2 is separated into two segments with a 70 amino acid long kinase insert region. Upon binding VEGFA and subsequent dimerization, VEGFR-2 is autophosphoryalted at the carboxy terminal tail and kinase insert region. Six tyrosine residues of VEGFR2 are autophosphorylated (see Fig.1[26]). within the activation loop of VEGFR2 leads to increased kinase activity[27]. (PDB code 3c7q).
Sorafenib inhibits cellular signaling by targeting several different receptor tyrosine kinases (RTKs) including receptors for platelet-derived growth factor (PDGFRs) and vascular endothelial growth factor receptors (VEGFR). PDGFR and VEGFR play crucial roles in both tumor angiogenesis and cellular proliferation. Sorafenib binds the ATP binding site of PDGFR & VEGFR, peventing the receptor kinase from binding ATP and phosphorylating their respective tyrosine target residues. Inhibition of PDGFR and VEGFR results in reduced tumor vascularization and cancer cell death. Sorafenib is also an inhibitor of KIT, a cytokine receptor inhibitor. Mutations of the KIT gene, often resulting in overexpression, are associated with cancerous tumors.[28] The KIT protein is at equilibrium between two predominant confirmations, the active conformation and the autoinhibited inactive conformation. In its active conformation, KIT binds to stem cell factors, upon which KIT dimerizes and transmits second messenger signals ultimately resulting in cell survival and proliferation. In its inactive conformation, the "DFG Triad" of KIT, residues Asp 810, Phe 811, Gly 812, is in the "out" position, with Phe 811 occupying the ATP binding site, preventing phosphorylation and signaling. The , is a good model for KIT as it shares numerous structural homologies, including conformations. Sorafenib inhibits p38 in an identical manner as it does KIT, by preferentially binding and stabilizing the autoinhibited inactive conformation of p38. using residues Glu 71, Leu 74, Val 83, Ile 166, His 148, Ile 84, Leu 167, Thr 106, His 107, Met 109, locking the inhibitor in place and stabilizing the receptor in the inactive state.[29]
Sunitinib inhibits cellular signaling by targeting several different receptor tyrosine kinases (RTKs) including receptors for platelet-derived growth factor (PDGFRs) and vascular endothelial growth factor receptors (VEGFR). PDGFR and VEGFR play crucial roles in both tumor angiogenesis and cellular proliferation. Sunitinib binds at the ATP binding site of PDGFR & VEGFR, peventing the receptor kinase from binding ATP and phosphorylating their respective tyrosine target residues. Inhibition of PDGFR and VEGFR results in reduced tumor vascularization and cancer cell death. Sunitinib is also an inhibitor of KIT, a cytokine receptor inhibitor. Mutations of the KIT gene, often resulting in overexpression are associated with most gastrointestinal stromal tumors.[30] is at equilibrium between two predominant confirmations, the active conformation and the autoinhibited inactive conformation. In its active conformation, KIT binds to stem cell factors, upon which KIT dimerizes and transmits second messenger signals ultimately resulting in cell survival and proliferation. In its inactive conformation, the "DFG Triad" of KIT, , is in the "out" position, with Phe 811 occupying the ATP binding site, preventing phosphorylation and signaling. by preferentially binding and stabilizing the autoinhibited inactive conformation of KIT (IC50 for Sunitinib is 40nM for inactive conformation and 21,000nM for active conformation). KIT binds Sunitinib using residues Lys 809, Val 603, Ala 621, Tyr 672, Cys 673, Leu 595, Cys 674, Gly 676, Leu 799, Glu 671 & Thr 670, locking the inhibitor in place and stabilizing the receptor in the inactive state.[31]
See also Bevacizumab.
See also Receptor
