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 -p- on tyrosine residue

tyrosine kinase domain -~50 RTK have been identified
-Has a single transmembrane spanning receptor -has 1 or 2 polypeptide chains
protein -has intrinsic kinase activity
- Largest family of enz linked receptors Ex: EGF,PDGF, FGF,IGF.
-monomers initially

STRUCTURE FUNCTION
Extracellular polypeptide ligand binding domain Regulates cell growth, differentiation, migration,
glycosylated (promotes specific, high affinity metabolism, proliferation, apoptosis, and
binding of ligand to the receptor) transcription
Single pass Trans membrane helix-hydrophobic
Intracellular catalytic kinase domain

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, protein kinase B (PKB/Akt),

growth factor receptor binding protein (Grb2), phosphatidylinositol 3-kinase (PIK3),
rapidly accelerated fibro sarcoma (Raf), p21-activated kinase kinase (PAK).
MAP kinase/ERK kinase (MEK),
extra cellular signal-regulated kinase (ERK),

RAS This protein was first identified as the oncogenic

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.
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 virus A and B), that are most often encountered in
dependent upon several posttranslational human cancers. Ras has a sequence comprising
modifications of a C-terminal “CAAX” motif such as 188–189 amino acids, with a cysteine residue,
farnesylation, Proteolysis, to increase its positioned four amino acids from the C-terminus
hydrophobicity, and promote membrane that requires addition of a farnesyl group (a 15-
association. carbon 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
RAS ISOFORM : LOCATION, SIGNALLING AND correct posttranslational processing of p21ras.
BIOLOGICAL ACTIVITIES Without these groups, the Ras proteins do not
Ras proto-oncogenes encode four highly associate with the cell membrane and are found to
conserved 21 Kda protein (p21ras). They are N-ras be inactive.
(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 GTPase-
activating 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 14-
3-3 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 14-
3-3 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 C-
Raf, 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 of malignant melanomas, carcinomas of ovary,

B-Raf is mutated in about 8% of human cancer. colon and thyroid. Over 30 single site point
Somatic B-Raf mutations are seen in 60% - 70% 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.

SORAFENIB (BAY 43-9006): SMALL

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 C-
Raf.
Sorafenib is found to reduce the basal
phosphorylation of MAPK pathway in human
cancer, melanoma, pancreatic cancer and colon
cancer.
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 B-
Raf 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.

DUAL MECHANISM OF SORAFENIB 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.
Sorafenib has moved into phase II and III clinical trials. paclitaxel in melanoma.
Recently sorafenibs are combined in vivo with
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

RAF DESTABILIZER Two novel Raf inhibitors CI-1040 (PD 184352)

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.
and PD 0325901 are currently in clinical trials. L-
779450, 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 c-
Raf. 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 pre-
clinical 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.