Kinases and cancer Flashcards
what are the hallmarks of cancer?
- Self sufficiency in growth signals
- Insensitivity to antigrowth signals
- Evasion of apoptosis
- Limitless replicative potential
- Tissue invasion and metastasis
- Sustained angiogenesis
what percentage of the population will develop cancer?
half
what are ‘conventional’ cancer therapies?
- Targeting generic properties of cancer cells (such as rapid proliferation)(post war medicine)(stlll the major way of treating)
o Cytotoxic agents, radiotherapy (combining them in an attempt to kill cells via dna damage to these rapidly dividing cell (more susceptible to radiation due to the replication))
o Surgery (if solid and no invasive (curative))
what are ‘modern’ cancer therapies?
- Targeting specific properties of cancer cells such as specific receptors or key proteins that are dysregulated (based on deep molecular understanding of the mechanisms that drive these cells)
o Monoclonal antibodies, kinase inhibitors (small molecules)
what types of protein control cell growth and proliferation?
7
often mutated in cancer
I. signalling molecules
II. signal receptors & intracellular receptors
III. intracellular transducers
IV. transcription factors
V. apoptotic proteins
VI. cell cycle control proteins
VII. DNA repair proteins
what mutations can convert proto-oncogenes into oncogenes?
- Gain of function mutations convert proto-oncogenes into oncogenes (all included in this lecture are)
o Point mutations: Single base pair change leaving protein constitutively active
o Chromosomal translocation: Results in hybrid gene with uncontrolled activity, Growth regulatory protein under control of different promoter causing inappropriate expression of the gene
o Amplification: Multiple DNA copies causes overproduction of respective protein
what types of kinases are there?
- Receptor tyrosine kinases eg EGF/ERB
- Cytosolic kinases eg Abl, src
- Nuclear kinases eg Jun Fos
how many kinases are there in the human genome?
512 kinases in the human genome
how can receptor tyrosine kinases be stimulated?
Multiple inputs – normally stimulated by EGF or related molecules, binding starts phosphorylation cascade eg MAPK, Pi3k, Mtor src or stats.
Is not stimulated if egf isn’t around.
Stimulation can occur by paracrine or autocrine
what is paracrine ErbB stimulation?
ERB ligands release from stromal cells and then binding their receptor
what is autocrine ErbB stimulation?
ER, GPCR, FZD stimulation activates metalloproteinase cleaving Pro-ERBB ligands (so they can dissociate and activate their receptor)
ER = oestrogen receptor, FZD = frizzled
breifly describe normal EGFR/ ErbB signalling
Ligand induce dimerization on the outside triggers increases cytosolic kinase activation i.e. self and cross-phosphorylation of Tyr residues. Depending on the receptor type and phosphorylation sites different intracellular signalling pathways are being activated.
ErbB receptors are abberrantly regulated in a wide range of cancers, what are the two basic ides for intervention?
- Inhibit dimerisation of EGF/ErbB receptors to suppress proliferation signal
- Inhibit kinase activity using small molecule tyrosine kinase domain inhibitors
what are the other names for ErbB1 and ErbB2?
ErbB1 = EGFR
ErbB2 = HER2
how many different types of ErbB receptors and ligands are there?
8 structurally related ligands signal via 4 structurally related EGF/ERB receptors
which ErbB receptors can forms homodimers or heterodimers?
EGFR/ErbB1 can form a homodimer with itself and a heterodimer with ErbB2
ErbB2 can form heterodimers with ErbB1, ErbB3 and ErbB4 (but NOT a homodimer)
ErbB3 can form a homodimer with itself and a heterodimer with ErbB2
ErbB4 can form a homodimer with itself and a heterodimer with ErbB2
ErbB1 doesnt interact with ErbB4
why is ErbB2 a primer target for therapy?
Erb2 is shared between all four of these receptors
It can form heterodimers with ErbB1, ErbB3 and ErbB4
how many ectodomains are there in the ErbB receptors?
4
describe the orientation of the ErbB (1, 3, 4) receptor when there is no ligand present
without ligand the ectodomain falls back on itself and creates an interface between domain 2 and 4
In this state the receptor is not able to dimerise and activate downstream
describe the molecular events that occur to ErbB (1, 3, 4) when a ligand comes along
when EGF comes along it binds between domains 1 and 3 causing a conformational/structural rearrangement at a pivot point between the two
Domain 2 is now exposed (with a beta strand extrusion) which then forms the dimer wiht another EGFR receptor dirving their intracellular domains in proximity so they begin to (auto)phosphorylate at a higher rate and then cross phosphorylating between the two to become the active complex
describe the similarities and differences between the confromations of ErbB receptors
- EGFR, ErbB3 and ErbB4 ectodomain undergo ligand induced reorganization making receptors dimersation competent. (erb2 doesn’t)
- Conformational regulation of ErbB3 and ErbB4 ectodomain dimerisation is similar to EGFR
ErbB2 ectodomain is dimersation competent in the absence of ligand
what is unqiue about the structure of ErbB2?
ErbB2 and EGFR:EGF complex have similar conformation
ErbB2 ectodomain is dimersation competent in the absence of ligand
is already in conformation to interact with ligand
It doesn’t need conformational change to bind and dimerise (this is why it does not form a homodimer)
We lose ability to play with egf binding but we can still block dimerisation
why doesnt ErbB2 form a homodimer?
ErbB2 ectodomain is dimersation competent in the absence of ligand
It doesn’t need conformational change to bind and dimerise (this is why it does not form a homodimer)
describe the working model for Erb3/2 homo and heterodimerisation
ErbB3 needs ligand to get upright and into a dimerization competent state then ErbB2 is brought into close proximity and cross phosphorylation ensues which activates the complex
ErbB2 is largely monomeric and inactive
what is the downstream signalling pathway of Erb2?
MAPK
what is the downstream signalling pathway of ErbB3?
PI3K-AKT
what two ways can we target EGFR/ErbB?
antibodies and small molecular inhibitors
give two ways antibodies can kill their target cell?
Complement binding: CDC complement dependent cytotoxicity
Fc receptor binding: ADCC antibody dependent cellular cytotxicity or phagocytosis
give five examples of antibodies aginst ErbB receptors?
- trastuzumab
- pertuzumab
- ceetuximab
- matuzumab
- pantumumab
what is the mechanism of action of Trastuzumab (herceptin)?
Trastuzumab (Herceptin) binds to the juxtamembrane region of Erb2 blocks proteolytic cleavage of the Erb2 ectodomain, avoiding remaining active kinase also antibody-dependent cellular cytotoxicity. Binds to the site close to the membrane, position type 4 domain.
This works by suppressing the receptor shedding (receptors are being shed leaving truncated version that can activate and become proliferatively active becoming lose cannons (by keeping 2 ectodomain suppresses the proliferative signal)
what is the mechanism of action of Pertuzumab?
binds directly to the Erb2 dimerization arm and blocks both dimerization and activation (domain two protrusion, neatly covers it to inhibit dimerization)
what is the mechanism of action of Certiximab?
Cetuximab (Erbitux) and humanized anti-EGFR antibody IMC-11F8 competes with ligand for binding to EGFR
using a dual mechanism of EGFR inhibition –
a) blocks the ligand binding site on EGFR domain III (egf bind between 3 and 1)
b) (stabilises) steric inhibition of extended, active like EGFR conformation.
The receptor is kept in the off state – cant interact with ligand or go into active state
what is the mechanism of action of matuzumab?
binds to domain III of EGFR non-overlapping site of EGF
– Matuzumab does not completely compete with EGF for binding to EGFR. But it does reduce the apparent affinity of EGF for EGFR.
– Matuzumab interferes with formation of active-like EGFR (keeps it in inactive state)
– In the presence of Matuzumab, EGF could only contact domain III (or domain I), and its affinity for EGFR would be reduced
Cetuximab and Matuzumab do not compete for binding to EGFR, hence ….
Cetuximab and Matuzumab do not compete for binding to EGFR, hence potential clinical benefit when used in combination therapy
why is it important to have different drugs that work via mutliple routes of inhibition
These put evolutionary pressure on the cancer so it finds escape mutants – proliferating cells that don’t bind to these antibodies – creating a resistance to the therapy so it no longer function as effectively
so then you can turn to another therapy that uses a different route that resistance hasnt been developed to yet
what is one limitation of trastuzumb ?
Preclinical results have shown that ErbB ligands can circumvent trastuzumab’ s ability to block downstream signalling and proliferation of ErbB2-overexpressing tumour cells.
Possible explanation: signalling competent heterodimers can still form
Even in presence of the mAb then you may still be able to get signalling competence
what element is needed for the phosphorylation by ErbBs to occur?
Magnesium
what are the two steps of kinase function in ErbBs?
1) autophosphorylation
2) target phosphorylation
describe the overal molecular structure of receptor kinases (ErbB )
ca 300 amino acids
Two lobes connected by hinge region:
- N-terminal lobe 5 stranded b-sheet and a single helix aC
- and larger C-terminal lobe mainly ahelical
ATP binding site located in the cleft between N- and C-lobe
what residues in ErbBs are important for catalysis?
Residues important for catalysis are located on both lobes
N-lobe: P loop (gly rich or nucleotide binding loop) β strand 3, and helix C (good for orientation of the active inactive state)
C-lobe: catalytic loop (β strands 6 and 7)
and the A-loop (activation loop) (contain DFG motif at the beginning of the loop, conformation changes dramatically between inactive and active) about 20 residues located between β strand 8 and helix α EF)
how does the A loop act as a pseudo substrate?
in the inactive state receptor kinases are in a diffferent conformation and such the A loop binds back into the receptor and sits where the ligand would usually bind blcoking access for the substrate
describe the different in the oritentation of the A loop between the active and inactive state of receptor tyrosine kinases
In the active state the A loop is protruding away from the molecule, ((dfg at beginning) the aspartic acid is important for aligning with the tertiary phosphate) phosphorylation has already occurred on tyrosine rings which leads to activation loop that goes away from the kinase
In the inactive state the a loop is in a different confromation, it is binding back into the receptor itself, it acts assa pseudo substrate so the a loop sits on the site where a ligand you usually bind
other changes do occur eg in the inactive state Helix c has diff orientation, at a different angle
Dfg motif is turned out in inactive state
typical mode of kinase autophosphorylation occurs by what mechanism?
o in the inactive state the activation loop is acting as a pseudo substrate.
when the ligand binds then
o Phosphorylation of Tyr/Ser/Thr residues in activation loop causes a significant rearrangement of the activation loop allowing subsequent substrate phosphorylation.
typical mode of kinase autophosphorylation occurs by what mechanism?
o in the inactive state the activation loop is acting as a pseudo substrate.
when the ligand binds then
o Phosphorylation of Tyr/Ser/Thr residues in activation loop causes a significant rearrangement of the activation loop allowing subsequent substrate phosphorylation.
what was observed when EGFR was compared to pY-IRK and IRK?
(ligand bound and ligand free)
Activation loop in EGFRK (unbound/inactive) structure similar to pY-IRK (active/ ligand bound)
