Receptor Tyrosine Kinase

RECEPTOR TYROSINE KINASE Transmits information from a factor outside a cell to the interior of a cell without requiring that factor to cross the cell membrane -Cell surface receptor that has intracellular tyrosine kinase domain -Has a single transmembrane spanning receptor protein - Largest family of enz linked receptors -monomers initially

-p- on tyrosine residue -~50 RTK have been identified -has 1 or 2 polypeptide chains -has intrinsic kinase activity Ex: EGF,PDGF, FGF,IGF.

STRUCTURE Extracellular polypeptide ligand binding domain glycosylated (promotes specific, high affinity binding of ligand to the receptor) Single pass Trans membrane helix-hydrophobic Intracellular catalytic kinase domain

FUNCTION Regulates cell growth, differentiation, migration, metabolism, proliferation, apoptosis, and transcription

MECHANISM: R (monomer) –L (gf) and R bind ---- R (dimerisation by non covalent binding) and R -Cytoplasmic domain auto p-this Stimulate kinase activity (catalytic domain)- auto p- and p- other molecules.on auto p-,receptor recruits CYTOPLASMIC SH2 prt (src homology domain) .thus activates several pathways at the same time. The transmission of a signal from the membrane to nucleus for activation of transcription can occur by multiple pathways EX: Ras-raf - mapk,PIK3 pathway. Initiated by receptor tyrosine kinase and G-protein-coupled receptors followed by transmission of signals by proteins that are regulated by kinases and phosphatases. In turn, these activated signalling elements phosphorylate amino acid residues on downstream signalling proteins. Proto-oncogenes that are important cell regulatory genes encodes proteins that function in the signal transduction pathways controlling normal cell proliferation while oncogenes are abnormally expressed or mutated resulting in abnormal cell proliferation and tumour development.

The central elements in the MAPK pathway are a family of protein – serine/threonine kinases such as Ras, growth factor receptor binding protein (Grb2), rapidly accelerated fibro sarcoma (Raf), MAP kinase/ERK kinase (MEK), extra cellular signal-regulated kinase (ERK), RAS Ras genes are small (21 to 24 Kda) Monomeric guanine nucleotide binding proteins Function in transduction of mitogenic signals from a variety of growth factor receptors.

protein kinase B (PKB/Akt), phosphatidylinositol 3-kinase (PIK3), p21-activated kinase kinase (PAK).

This protein was first identified as the oncogenic proteins of tumour viruses that cause sarcoma in rats. Ras proteins are prototypes of large family of ~50 related proteins, called small GTP –binding proteins.

Ras is synthesized in the cytoplasm and is dependent upon several posttranslational modifications of a C-terminal “CAAX” motif such as farnesylation, Proteolysis, to increase its hydrophobicity, and promote membrane association.

virus A and B), that are most often encountered in human cancers. Ras has a sequence comprising 188–189 amino acids, with a cysteine residue, positioned four amino acids from the C-terminus that requires addition of a farnesyl group (a 15carbon terpenoid). They also carry an S-palmitoyl (C16 fatty acid) group. The C-terminal amino acids (186-189) form a conserved CAAX box required for correct posttranslational processing of p21ras. Without these groups, the Ras proteins do not associate with the cell membrane and are found to be inactive.

RAS ISOFORM : LOCATION, SIGNALLING AND BIOLOGICAL ACTIVITIES Ras proto-oncogenes encode four highly conserved 21 Kda protein (p21ras). They are N-ras (neuroblastoma cell line), H-ras (Harvey murine sarcoma virus), and K-ras (Kirsten murine sarcoma ACTIVATION OF RAS PATHWAY The binding of epidermal growth factor (EGF) induces receptor dimerisation and autophosphorylation (P) on tyrosine residues on the cytoplasmic side of the cell membrane. These phosphotyrosines activates the small G-protein Ras by stimulating the exchange of guanosine diphosphate (GDP) for guanosine triphosphatases (GTP). Tyrosine Phosphorylation of receptors creates a binding site for a small protein called Grb2 SH2 domain. Grb2 associated with another small protein called Son of sevenless through a binding domain (SH3) recognises proline-rich regions on Sos.

Once activated, this receptor-GRB2-Sos protein complex catalyses the exchange of GTP for GDP resulting in formation of the active Ras-GTP complex. Activation of the Ras GTP complex is then terminated by GTP-hydrolysis, which is stimulated by the interaction of Ras-GTP with GTPase-activating proteins. This exchange elicits a conformational change in Ras. This then recruits a kinase called Raf, from the cytosol to the plasma membrane where Raf can be activated by other signals. This ultimately activates a promoter gene within the cell nucleus where transcription of DNA leading to messenger RNA (mRNA) production can then proceed, and these codes for the production of enzymes associated with cell growth and differentiation. Thus Ras proteins are controlled by GTP-GDP binding. RAS ONCOGENES The ras oncogenes are not present in normal cells; they are produced in tumour cells as a result of mutation that occurs during tumour development. The Ras oncogenes differ from their proto-oncogenes by point mutation (single amino acid substitution at position Gly12/Gly13 or Gln61). Ras oncogenic activation by point mutation results in inability to hydrolyse the GTP in presence or absence of GTPaseactivating protein (GAP) resulting in continuous expression of Ras favouring malignancy. Mutated Ras genes are found in about 15% of all human cancers including 25% of lung cancer, 50% of colon cancer, and 90% of pancreatic cancers. Mutation converts normal Ras genes to oncogenes in human cancers and inhibits GTP hydrolysis by the Ras protein. Thus mutated Ras oncogenes maintain the Ras protein continuously in the active GTP-bound conformation. Then, GAP stimulates hydrolysis of bound GTP by normal Ras. Due to decrease in intracellular GTPase activity, the oncogenic Ras proteins remain in the active GTP bound state and drive unregulated cell proliferation. Thus it could be concluded that Ras is not only capable of inducing abnormal growth but also appears to be required for the normal cells proliferation, even in the absence of growth factors that would be required to activate Ras and signal proliferation of normal cells.Ras is mutated frequently in human cancers. Ras proteins are targeted to plasma membrane by the posttranslational modification by the attachment of lipids to the polypeptide chains. Thus plasma

membrane associated Ras proteins involved in the control of cell growth and differentiation are modified and are responsible for the uncontrolled growth of human cancers. Such modifications anchor the proteins to the plasma membrane and interact with the hydrophobic lipid. This type of modification is called farnesylation, in which 15-carbon hydrophobic farnesyl isoprenyl tail is added to carboxyl terminus of Ras, catalysed by farnesyl transferase. This step is followed by proteolytic removal of the cysteine residue at the carboxyl-terminus and methylation (addition of methyl group) of cysteine to the carboxyl group of the C-terminal cysteine residue. TARGTING RAS PATHWAY FOR CANCER RAS PATHWAY INHIBITORS THERAPY Other inhibitors of Ras are geranylgeranyl Targeting Ras as a therapeutic strategy raises transferase-I (GGTase-I), isoprenylcysteine the possibility of developing drugs that might carboxylmethyltransferase (ICMTase-I), statins, selectively act against cancer cells. Thus, it has bisphosphonates, small inhibitory RNA (SiRNA), attracted considerable interest as potential drug combination of inhibitors such as gefitinib, targets. Inhibitors of enzyme farnesyl transferase erlotinib, bevacizumab, lonafarnib are also used are found not only to inhibit Ras membrane to silence Ras expression in human cancer cell localization and function, but also display lines and the effects of Ras silencing on considerable selectivity in their action against proliferation, apoptosis, and tumour growth are tumour cells expressing oncogenic Ras proteins. assessed. The potential of these drugs in When drugs such as the farnesyl transferase treating human cancers that involve in ras inhibitors block farnesylation, Ras is unable to oncogenes now awaits evaluation in clinical anchor to the cell membrane and its function is trials. impaired. RAF KINASE The Raf Serine/ threonine specific protein kinase signalling pathway has been highly conserved throughout evolution, and activation of the Raf protein kinase is considered to be a primary event in the Ras signalling pathway. This Raf signalling pathway promotes cell survival, proliferation and apoptosis. It was first identified as oncogenes in retroviruses that are causative vectors in tumours in mice and chicken. There are 3 Raf genes. A-Raf, B-Raf and c-Raf. A-RAF, B-RAF AND C-RAF A-Raf (68 Kda) located on Xp11 is the weakest activator of MEK, and can only activate MEK1 but not MEK2 undergoes localization to the mitochondria which links with the regulation of apoptosis and is over expressed in urogenetial tissues (Kidney and ovary). So far, no mutations in A-Raf has been found in human cancers. Alternatively spliced B-Raf (94 Kda) located on 7q32 is the strongest Raf kinase that induces MEK activity that is over expressed in neural, testicular and haemopoietic tissues. Recent studies have shown 30 single-site missense activating mutations within the B-Raf kinase domain in human cancers. This has primarily directed to consider B-Raf as a potential therapeutic target. The majority of the somatic mutations of B-Raf are found in two regions: the glycine-rich loop of the kinase domain, and within or adjacent to the activation segment. A Glu for Val substitution at residue 600 (V600E, previously designated V599E) is seen in 90% of B-Raf mutations, resulting in constitutive kinase activity, which can transform NIH3T3 cells. B-Raf mutations have been found at inhibitory Akt phosphorylation sites in the CR2 domain. Recent evidence shows that presence of B-Raf mutations may resolve sensitivity to drugs that target the ERK pathway at the level of Raf kinase. C-Raf (Raf-1, 74 Kda) is a mitochondrial protein located on 3p25, which undergoes localization to the mitochondria. It is ubiquitously expressed in adult tissues. Mutations in Raf-1 have not been detected in human cancers STRUCTURE AND FUNCTION OF RAF KINASE Raf protein consists of 3 regions: amino terminus (regulatory domain), activation loop and carboxyl terminus (catalytic domain). All 3 Raf genes have 3 conserved regions, and several regulatory phosphorylation sites: CR1 (adjacent to N’), CR2, CR3 (adjacent to C’). The GTP-bound form of ras directly interacts with N-terminal region of C-Raf. This binding localizes Raf to the

plasma membrane. Initial process of Ras activation requires active GTP binding with Ras binding domain of Raf CR1 while CR2 rich in proline and threonine residues, is involved in protein-protein interaction, negative regulation of Raf activity by Akt or protein kinase A phosphorylation at serine (S) residue S259 and in localization of Raf and the catalytic domain of

Raf, CR3 involves in positive regulation of Raf Phosphorylation on S338, tyrosine (Y) Y340 and Y341.

REGULATION OF RAF KINASES All Raf kinases are activated by the Ras small GTPase, regulatory phosphorylation events, scaffolding proteins (kinase suppressor of Ras and MEK-1), adaptor proteins (Bcl-2-asociated athanogene-1), Chaperone proteins (Hsp90 and Hsp70), substrates (Retinoblastoma protein) and lipids. The combination of all these events leads to the proper activation of Raf. Hence, the complexity of Raf activation depends on different isoforms of Raf that combine with common unique mechanisms to regulate Raf kinase activity. Raf kinase activation is a complex multistep process initiated with the recruitment of inactive Raf (complexed with 14-3-3 and heat shock proteins) from the cytosol to the plasma membrane by activated Ras. Ras, binds to Raf at the Ras-binding domain and cysteine-rich domains of the CR1 region. Once recruited Raf undergoes modifications before getting actived. A-Raf and C-Raf normally exists in an inactive state. Raf is inactivate due to phosphorylation at residues S259 and S621 that binds with the highly conserved chaperonin protein 14-3-3, which maintains Raf in an inactive conformation. To become activated Raf requires to undergo dephosphorylation of S259 and S621 which dissociates 143-3 from Raf, and then phosphorylation at residues S338, Y340, and Y341.S259 is also found to be the site of inhibitory phosphorylation by Protein kinase B/Akt, PKA, Serum glcocorticoid-inducible kinase. Recent studies have proved that the post-translational modification of Ras p21 is necessary for C-Raf activation by v-ras p21 in Sf9 cells. Various processes such as protein –protein and protein –lipid interactions, regulate c-Raf. C-Raf controls 14-3-3 binding sites: S259, S621, CRD while B-Raf controls 143-3 binding sites: S429, S426, S728. B-Raf is constitutively phosphorylated upon translocation to the plasma membrane on serine (S445), which corresponds to a regulated phosphorylation site (S338) on CRaf, and because a regulatory tyrosine residue (Y341) is replaced by an aspartic acid residue (D448). Thus activation of B-raf induces a downstream signal transduction cascade. RAF ONCOGENES Ras genes can be converted to oncogenes by deletion that leads to loss of amino-terminal regulatory domain of Raf protein. Constitutive activation of Raf mutation observed in melanoma, haematopoietic cancer, thyroid, kidney, liver, larynx, biliary tract and breast cancer results in point mutation, deletion, amplification, and rearrangement of Raf.

B-RAF MUTATION B-Raf is mutated in about 8% of human cancer. Somatic B-Raf mutations are seen in 60% - 70%

of malignant melanomas, carcinomas of ovary, colon and thyroid. Over 30 single site point mutation encoding amino acids within the kinase

domain of B-Raf gene are identified to cause Thymidine to adenine transversion (1796 position in exon 11/15) encoding valine to glutamic acid substitution at amino acid 599. Basal kinase activity of v599EB-Raf is 12.5 fold higher than wild type B-Raf. Oncogenic b-raf stimulates proliferation, constitutive signalling, ERK pathway (without Ras activation and transforms NIH3T3 cells) and survival in cancer. C-RAF MUTATION

C-Raf is mutated in about 0.7 % of human cancer. Constitutively active C-Raf has been associated with site-specific C-Raf mutation. Gene rearrangements of C-Raf mutations are found in human cancer patients with NSCLC and T-Cell lymphoma. A structurally aberrant C-Raf protein, reduced in its amino terminal regulatory domain has been identified in Kidney, lung, liver and pancreatic carcinoma; Tissue and bone sarcoma; CNS malignancies.

TARGTING RAF PATHWAY FOR CANCER THERAPY Drugs targeting the MAPK pathway at the level of Raf are useful as Raf is one of the activator of the ERK pathway, whereas other upstream targets such as the growth factor ligands, receptor tyrosine kinases or even Ras, have many other potential effectors. Raf has been mainly focussed as new-drug-development efforts due to the high percentage of human tumours harbouring oncogenic raf. Most effectors have been directed to C-Raf than B-Raf. Thus, agents targeting the Raf proteins on the whole or C-Raf exclusively have been widely developed in many pre-clinical and clinical trials. Various therapeutic agents have been developed to inhibit Raf production and activation. They are antisense oligonucleotides (ASONs), Small molecule kinase inhibitors, and Dominant interfering DNA constructs. Other therapeutic agents include chaperones, which destabilises Raf indirectly, and histone deacetylase inhibitors, that reduces Raf expression. ISIS 5132: AN ASON INHIBITOR OF C-RAF ASONS are short synthetic oligonucleotides, complementary to Raf mRNA. Intra cellularly, these oligonucleotides hybridise with their cognate mRNA and results in degradation of RNase-H mediated complex. Moreover, ASONS are capable of inhibiting translation, which reduces the synthesis of the encoded protein. ISIS 5132 is 20 base phosphorothioate oligonucleotide and a highly active suppressor of c-raf mRNA both in vitro and in vivo. It hybridises to the 3‘untranslated region of the human C-Raf mRNA. Binding of ISIS 5132 to C-Raf mRNA appears to encourage RNase-H mediated C-Raf mRNA degradation. Thus, reduces the synthesis of CRaf.

Sorafenib is found to reduce the basal phosphorylation of MAPK pathway in human cancer, melanoma, pancreatic cancer and colon cancer. DUAL MECHANISM OF SORAFENIB

SORAFENIB (BAY 43-9006): SMALL MOLECULE INHIBITOR OF RAF KINASE Sorafenib is one of the most potential and promising agents of the class of Raf kinase inhibitors. It is an orally available bi-aryl urea (4{4-[3-(4-chloro3-trifluoromethyl-phenyl)-ureido]phenoxyl}-pyridine-2-carboxylic acidmethylamide-4-methylbenzene-sulfonate). It is a competitive inhibitor of ATP binding in the catalytic binding domains of C-Raf, wild type BRaf and mutant B-Raf. Biochemical studies have shown that sorafenib inhibits RTKs involved in tumour progression and angiogenesis while MEK1, ERK1, erbB1, erbB2 pathways are not inhibited.

Recent evidence shows that Sorafenib inhibits phosphorylation of several pro-angiogenic RTKs and reduces tumour neovascularisation significantly Consequently the researchers found that sorafenib is capable of targeting both tumour progression by blocking cellular proliferation that is dependent on activation of MAPK pathway and tumour angiogenesis through VEGFR2, 3 and /or PDGFR-ß. By inhibiting Ra kinase, Sorafenib blocks the RAF/MEK/ERK signalling pathway that is activated by the Ras gene to trigger cel proliferation.

At present, drugs are designed to prevent signals that activate the Ras, while sorafenib

interrupts downstream signalling enzyme from Ras, namely Raf kinase. This data suggests that sorafenib has the potential to be effective against cancers caused by abnormal external activation of Ras, as well as abnormal stimulation of growth due to mutations in the Ras gene and mutations in Raf, making sorafenib a possible anticancer application.

gemcitibine, cisplatin, irinotecan, vinorelbine, paclitaxel, or gefitinib. Currently, combination trails are underway using sorafenib (BAY 43-9006) with carboplatin, paclitaxel and other chemotherapeutic agents, including a Phase III trial for advanced melanoma. Combination drug therapy to inhibit Raf: BAY 43-9006 and gemcitibine in advanced ovarian and pancreatic cancers; BAY 43-9006 and doxorubicin in hepatocellular carcinoma; BAY 43-9006 and oxaliplatin in colorectal cancer; BAY 43-9006, carboplatin, and Sorafenib has moved into phase II and III clinical trials. paclitaxel in melanoma. Recently sorafenibs are combined in vivo with

RAF DESTABILIZER Geldanamycin is a benzoquinone ansamycin that binds to heat shock protein 90 (hsp90), disrupting the C-Raf hsp90 multimolecular complex, which leads to C-Raf destabilization and degradation via cellular proteolytic mechanisms such as the proteasome-mediated pathway. Phase I and II trials with 17 AAG, are less clinically toxic analogue of geldanamycin, and a phase I trial of the second-generation geldanamycin analogue, 17-DMAG, are currently ongoing.

Two novel Raf inhibitors CI-1040 (PD 184352) and PD 0325901 are currently in clinical trials. L779450, are more effective at inhibiting kinase activity of C-Raf and A-Raf than B-Raf. Phenol substituted oxindole derivative SB203580, inhibits Raf kinase in the low nanomolar range. Another small molecule Raf inhibitor, SB-590885 has been recently reported from GlaxoSmithKline. Radicicol, has inhibitory activity against a wide range of human tumour cell lines and xenografts. It also inhibits tumour growth by destabilizing and depleting the cRaf. O-carbamoylmethyloxime derivatives are found superior to radicicol from both mechanistic and pharmaceutical perspective.

OTHER PHARMOCOLOGIC INHIBITIORS OF RAF KINASE DOMINANT INTERFERING DNA CONSTRUCTS DNA constructs suggest therapeutic promise for the delivery of drugs. Moreover, targeting genes to specific blood vessels provide complementary approaches to induce the growth of new blood vessels in various disease states. These dominant interfering DNA constructs targets tumour cells with anti-RAF gene. This technique uses cationic lipid-based nanoparticles and αvβ3 integrin ligands. CONCLUSION Recognition of intracellular signalling cascades is important for the growth and survival of cancer cells and has led to the development of targeted cancer therapeutics that seeks blocking these signals. The mitogen-activated protein kinase (MAPK) pathway has a well-defined role in cancer biology and has been an important target in the development of targeted therapies. Raf is central to MAP kinase signalling and is frequently activated in cancer. Raf has been an attractive cancer target but lacks clinical efficacy that uses agents to target this protein has raised serious doubts about its therapeutic utility. Recent preclinical studies combining sorafenib with other agents to increase its efficacy are providing a mechanistic basis for selection of novel agent combinations that might be clinically effective.