Kinases and cancer Flashcards
(114 cards)
1
Q
what are the hallmarks of cancer?
A
- Self sufficiency in growth signals
- Insensitivity to antigrowth signals
- Evasion of apoptosis
- Limitless replicative potential
- Tissue invasion and metastasis
- Sustained angiogenesis
2
Q
what percentage of the population will develop cancer?
A
half
3
Q
what are ‘conventional’ cancer therapies?
A
- 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))
4
Q
what are ‘modern’ cancer therapies?
A
- 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)
5
Q
what types of protein control cell growth and proliferation?
7
often mutated in cancer
A
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
6
Q
what mutations can convert proto-oncogenes into oncogenes?
A
- 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
7
Q
what types of kinases are there?
A
- Receptor tyrosine kinases eg EGF/ERB
- Cytosolic kinases eg Abl, src
- Nuclear kinases eg Jun Fos
8
Q
how many kinases are there in the human genome?
A
512 kinases in the human genome
9
Q
how can receptor tyrosine kinases be stimulated?
A
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
10
Q
what is paracrine ErbB stimulation?
A
ERB ligands release from stromal cells and then binding their receptor
11
Q
what is autocrine ErbB stimulation?
A
ER, GPCR, FZD stimulation activates metalloproteinase cleaving Pro-ERBB ligands (so they can dissociate and activate their receptor)
ER = oestrogen receptor, FZD = frizzled
12
Q
breifly describe normal EGFR/ ErbB signalling
A
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.
13
Q
ErbB receptors are abberrantly regulated in a wide range of cancers, what are the two basic ides for intervention?
A
- Inhibit dimerisation of EGF/ErbB receptors to suppress proliferation signal
- Inhibit kinase activity using small molecule tyrosine kinase domain inhibitors
14
Q
what are the other names for ErbB1 and ErbB2?
A
ErbB1 = EGFR
ErbB2 = HER2
15
Q
how many different types of ErbB receptors and ligands are there?
A
8 structurally related ligands signal via 4 structurally related EGF/ERB receptors
16
Q
which ErbB receptors can forms homodimers or heterodimers?
A
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
17
Q
why is ErbB2 a primer target for therapy?
A
Erb2 is shared between all four of these receptors
It can form heterodimers with ErbB1, ErbB3 and ErbB4
18
Q
how many ectodomains are there in the ErbB receptors?
A
4
19
Q
describe the orientation of the ErbB (1, 3, 4) receptor when there is no ligand present
A
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
20
Q
describe the molecular events that occur to ErbB (1, 3, 4) when a ligand comes along
A
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
21
Q
describe the similarities and differences between the confromations of ErbB receptors
A
- 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
22
Q
what is unqiue about the structure of ErbB2?
A
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
23
Q
why doesnt ErbB2 form a homodimer?
A
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)
24
Q
describe the working model for Erb3/2 homo and heterodimerisation
A
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
25
what is the downstream signalling pathway of Erb2?
MAPK
26
what is the downstream signalling pathway of ErbB3?
PI3K-AKT
27
what two ways can we target EGFR/ErbB?
antibodies and small molecular inhibitors
28
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
29
give five examples of antibodies aginst ErbB receptors?
* trastuzumab
* pertuzumab
* ceetuximab
* matuzumab
* pantumumab
30
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)
31
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)
32
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
33
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
34
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
35
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
36
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
37
what element is needed for the phosphorylation by ErbBs to occur?
Magnesium
38
what are the two steps of kinase function in ErbBs?
1) autophosphorylation
2) target phosphorylation
39
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
40
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)
41
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
42
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
## Footnote
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
43
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.
43
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.
44
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)
45
If in the EGFRK the A loop is already our irrespective of activation state then how is activity controlled?
Is controlled through interaction with the N terminal domain
46
what was the recent observtaion made about EGFR kinase dimers
they are activated by self interaction with another EGFRK
The dimer you get is asymmetric – one is active one is inactive
47
describe the process of the asymmetric EGFR kinase dimer activation
In the absence of ligand, EGFR adopts a compact conformation in which a loop on domain II is buried (left).
Ligand binding promotes a domain rearrangement in which domains I and II rotate and expose the domain II loop.
The exposed domain II loop mediates dimerization of the extracellular regions, which leads to formation of an asymmetric dimer of the kinase regions, activation of the 'acceptor' kinase by a 'donor' kinase and transphosphorylation of the C-terminal tail region.
C-lobe interaction of the donor kinase interacts with N-lobe of the acceptor kinase, activating the acceptor kinase but not the donor kinase. When this happens the tail of the acceptor is phosphorylated and becomes active
| non signalling donor kinase, active acceptor kinase
48
describe the two stages of EGFR/ErbB activation
Inactive kinase with low catalytic state
– when substrate binds the receptor is triggered to self phosphorylate, the rate of phosphoylation increases which is when the target can come in to be phosphorylated at tyrosine residues so the target can now interact with others
49
at what places does ATP interact with EGFR/ErbB?
Two interaction sites (hydrophobic pocket1) on C-lobe and another on the N-lobe (hydrophobic pocket 2)
also allosteric site
50
why is ATP inhibtion so difficult?
there are Millimolars of atp in the cell (high) so competing is hard bcos it is so abundant and also is used by many enzymes
(designing an inhibitor needs to be very high affinity and extremely specific or the cells will be killed by off target effects)
51
how can we distinguish between the two type of ATP inhibitors for EGFR/ErbB?
they either target the active state or the inactive state
52
when/how does Iressa (gefitinib) target EGFR?
Gefitinib binds to EGFR at its highly important ATP binding activation site which is located at lysine 745 (bound by other aa aswell)
targeting it in the active conformation
Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated
53
when/how does Lapatinib target EGFR?
it binds the allosteric site when the receptor is in the inactive state
allosteric site is only available in the inactive state as the helix move to site and the DRG is move out
54
what is the significance of the set of cysteines around the active site of EGFR or other kinases?
* Kinases can be classified by where the cysteines are around the active site (goup 1 group 2 group3 group 4)
* Irreversible inhibitors using reactive kinase cysteine residues
55
what is staurosporine?
Staurosporine is a cell permeable alkaloid isolated from Streptomyces staurosporeus exhibiting anti-cancer activity. Staurosporine is a potent, non-selective inhibitor of protein kinases, including protein kinase C. This agent induces apoptosis
56
what is the mechanism of staurosporine?
it acts by forming covalent bonds with the cysteines (near the active site) enabling you to inhibit the activity
make suicide molecules where you have irreversible covalent bond formation between the inhibitor and the cysteines near the active site so you inhibit the complex entirely
57
how do escape mutants effect small moelcule inhibitors for EGFR/ErbB?
evolving mutations in active site that render the inhibitor ineffective
58
what is erlotinib?
| tarceva
Erlotinib (Tarceva)
Erlotinib is a type of targeted cancer drug that inhibits tyrosine kinase activity by binding to the ATP-binding pocket of EGFR,
It is a treatment for:
non small cell lung cancer (NSCLC) that has spread (advanced)
advanced pancreatic cancer – alongside the chemotherapy drug gemcitabine
59
describe the mechanism of dysregulation in receptor tyrosin ekinases
60
describe how ligand independent firing can occur in EGFR/ErbBs
61
what cancers have the BCR-ABL gene fusion?
The BCR-ABL fusion gene is found in most patients with chronic myelogenous leukemia (CML), and in some patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML)
62
chronic myelongenous leukemia account for what percentage of all leukemias?
* Accounts for ca 15% of all leukemias
63
in the US how common is CML?
1/500
64
what are the two phases of CML and how do they differ in the cells that are involved?
– Chronic phase (may last months to years)
* Elevated numbers of (largely mature) white blood cells (monitored at this stage bcos nothing really can be done until it gets into the acute stage)
– Acute phase
* Significantly increased number of largely immature white blood cells (at this stage you start treatment)
65
when was the first clear description of leukemia? by who?
| what was the phenotypic description?
1865 by Hugh Bennet and Rudolph Virchow
Phenotypic description at autopsy: enlarged spleen, severe aneamia and leucocytosis
66
what was discovered about chronic myelogenous leukemia in 1960?
1960 Discovery 2: Association with Philadelphia Chromosome
Then they observed those with CML had chr abnormalisty – chr22 v shot (Philadelphia bcos that was the uni) – hypothesising CML was caused by this abnormality
“The findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocyte leukemia”
67
what did Rowley discover in 1973?
1973 Discovery 3: Identification of BCR-ABL
Rowley found it came about by translocation – forming a fusion gene of BCR and ABL
Philadelphia Chromosome: reciprocal translocation between chromosome 9 and 22
68
CML originates in what cell type?
hematopoietic stem cells
(evidence from virology)
69
what three major findings were discovered in 1975-1980?
1975: Abelson murine leukaemia virus transforms NIH into lymphoid cells (effect of virus transforms cell lines into cancer)
1978-80: Ableson murine leukaemia virus encodes gag-abl ((pre)sequencing found this in the virus)
1980: Identification of ableson tyrosine kinase as oncogenic activity in GAG-ABL (this protein was responsible for the transformation)
70
how was virology involved in the identification of BCR-ABL?
observation that Abelson murine leukaemia virus transforms NIH into lymphoid cells (effect of virus transforms cell lines into cancer)
lead to the discvoery of the gene responsible
Ableson murine leukaemia virus encodes gag-abl ((pre)sequencing found this in the virus)
Identification of ableson tyrosine kinase as oncogenic activity in GAG-ABL (this protein was responsible for the transformation)
this is a version of BCR-ABL fusion gene that is the culprit in humans
71
when was the philadelphia chromosome characterised as BCR-ABL with abl tyrosine kinase activity?
1982-86
72
what is the primery structure of Abl tyrosine kinase?
N terminal has the kinase domain, and regulatory domains SH2 and SH3 with a Myristate group at the end (to localise to membranes). Autoinhibitory cap, SH2 kinase linker
(kinase self assembly domains (
C terminal contains localisation cues - including Nuclear localisationsignal, DNA bindingm and F actin binding. NUclear export signal, coil coil domain, PxxP motif
73
how do we know Abl has roles in the nuclear and cytoplasm?
| shuttles between the two in response
bcos it has nuclear localisation singals and dna binding as well as f actin binidng ability (located in the cytoplasm)
74
molecularly what does SH3 bind to ? (the domain)
poly proline binding domain: recognises PxxP motif
75
molecularly what does SH2 bind to?
phosphotyrosine recognition domain
76
how do we knwo Abl is a crucial gene?
* ABL knockout is embryonic lethal in mice
77
by default what state is Abl in?
* Basal state of ABL has low phosphorylation activity: by default ABL is in the “off-state”
78
the off state of abl is maintained by...
| breifly
– Self-interactions (cis)
– interactions with range of targets (trans)
79
describe the strcture of abl in the autoinhibited state
Essential features for maintaining the auto inhibited state:
* **association of SH3 with N-lobe** Via polyproline motif helix)(keeps kinase in a low activity state, maintains rigidity) (changes in protein on the N lobe can change the activity of the kinase)
* **Insertion of Myristol group in C-lobe **
80
breifly what is the route to ABL activation
Partial activation
1) unlatching: removal of myristyl group from C-lobe leads to partial activation
2) unclamping: displacement of SH3 from N-lobe
Full activation: Switching: phosporylation of Tyr-412 in activation loop & Tyr-245 in SH2- kinase linker
81
describe the route to ABL activation
N terminus can inserts the myristol group into the C terminus
If can remove the interactions that hold the N terminal region of the AbL kinase – Unlatching
Then need to stop the N lobe-SH3 domain interaction by providing a more favourable interaction (outcompete), eg a peptide sequence that has an SH3 binding domain and an SH2 recognising phosphotyrosine motif. If this is high enough affinity (eg Grb2), then can cause release of the self interactions of kinase domains with regulatory domains
Followed by an autophosphorylation event of Tyr245 in the linker between the kinase domain and the SH2 domain
and Tyr412 phosphorylation of the activation loop opening it up so the active site is open to bind substrate. and the kinase is active
82
the activation of Abl is similar to what other family of kinases and why?
The activation is similar to Src kinases
ABL tyrosine kinase core is shared with Src family kinases
83
how is viral abl different from human endogenous abl?
viral abl doesnt have the SH3 domain
in its place it has a GAG
(hence GAG-ABL)
84
what is the difference in structure between the fusion protein BCR-ABL and normal ABL
BCR -ABL has the BCR gene fragment in place of the myristate group at the N terminus
the leads to the difference in functionality
85
what is cis control in terms of abl regulation?
self interaction
86
describe the actiation of Src kinase family
SRC has a C terminus of phosphotyrosine that can interact with the SH2 domain and stabilise that.
Dephosphorylation of that residue Tyr527 and therefore weaken interaction wit the SH2 domain and then offer phosphotyrosine peptides that also have a PxxP to make high enough affinity ligands to dissociate that pari of domains and activate the kinase
After this get autophosphorylation on the activation loop, Tyr416 phosphorylation that results in fully activate Src and phosphorylates downstream targets
87
cellular fate of cells depend on ABL .____..
localisation
88
how can abl lead to cell survival or cell death
* ABL activity can lead to cell survival in response to growth factors concomitant with cytosolic localisation
* but also to cell death in response to DNA damage and oxidative stress concomitant with nuclear accumulation
89
abl activity is ____ dependent
abl activity is context dependent
90
why are kinases harder to target than receptor tyrosine kinases?
Kinases like ABL have multiple input signals, inhibitors, activators and outputs that can arise in multiple biological processes
Receptor tyrosine kinases are much simpler in that they receive an input from the outside, resulting in activation, substrate phsophorylation resultign in downstream signalling cascades. they only have one input type and then multiple outputs. they have an inherent advantage for therapy because of this simiplicity
91
what is the primary structure of BCR?
| domains?
DD dimerization domain,
cAMP: cyclic adenosine monophosphate kinase homologous domains
RHO-GEF: homologous to Rho guanidine nucleotide exchange factors well as dbl-like and pleckstrin homology (PH) domains.(PH domains interacting with phosphoinositides) (exchange factors are important in secondary messenger regulation)
CaLB: putative Calcium dependent lipid binding domain with activation function of Rac-GTPase (RAC-GAP)
92
BCR-ABL fusion can result in three different fusion protein? what are they? which is the most common?
breakpoints in BCR-ABL fusion leading to three fusion proteins.
P190
P210
P230
Most prevalent is P210 fusion withABL
93
what cancer signallign pathways is BCR-ABL involved in?
RAS (MAPK/ERK pathway)
PI3K
apoptotic (via BAD/AKT)
MYC (unknown linker)
cytoskeletal proteins
STAT
## Footnote
Ras-Raf-MEK-ERK = MAPK
94
What are the functions of the three main downstream pathways of BCR-ABL?
| that lead to the malignant phenotype
altered adhesion
mitogenic activation
inhibition of apoptosis
95
how does GAG-ABL lead to malignancy?
| viral abl
* GAG-ABL (v-ABL) lacks SH3 domain
– Suggest that lack of SH3-N-lobe interactions lead to constitutive activity
– Shown to be a controlling factor for activation
96
Molecularly how does BCR-ABL lead to malignancy?
* BCR ABL lacks myristate group
– Suggest that lack of myristate insertion in C-lobe leads to constitutive activity
– Doesn’t seem to have a direct effect – not enough to explain its oncogenic abilityies
* BCR domain mediates dimerisation and association effector proteins.- recruiter due to interaction sites
97
what are two options for inhibition of BCR-ABL?
BCR itself upregulates the kinase actvity of Abl to cause problem so inhibition of the kinase activity is
* ATP required for tyrosine phosphorylation activity - Tyrosine Kinase inhibitor (TKI)
* Alternatives: e.g substrate (Tyr) competitors
o Early attempts did not yield successful inhibitors
98
what are some of the challenges for TKIs?
| tyrosine kinase inhibitors (atp blocking)
* ATP is essential in many processes for cell survival
– Specificity problem:
a) Binding site of kinase the domains of ABL and BCR ABL have the same sequence. Effect of ABL inhibition? ( if you inhibit the ATP binding somain on bcr abl then you will also be targeting but abl aswelll)
b) 512 Kinases in the human genome (other kinases bind ATP so we would need specificty)
c) Significant numbers of ATP dependent proteins ( enzymes) other than kinases
– ATP is abundant (**2-5mM concentration in cells**) so need high affinity competitor
ATP inhibition is a nightmare in terms of chemistry and bio – need super high affinity and super specific affinity for it to be effective
98
what are some of the challenges for TKIs?
| tyrosine kinase inhibitors (atp blocking)
* ATP is essential in many processes for cell survival
– Specificity problem:
a) Binding site of kinase the domains of ABL and BCR ABL have the same sequence. Effect of ABL inhibition? ( if you inhibit the ATP binding somain on bcr abl then you will also be targeting but abl aswelll)
b) 512 Kinases in the human genome (other kinases bind ATP so we would need specificty)
c) Significant numbers of ATP dependent proteins ( enzymes) other than kinases
– ATP is abundant (**2-5mM concentration in cells**) so need high affinity competitor
ATP inhibition is a nightmare in terms of chemistry and bio – need super high affinity and super specific affinity for it to be effective
99
what is Imatinib?
a type of cancer growth blocker, tyrosine kinase inhibitor for ABL (and related proteins, including BCR-ABL)
also known as STI-571 or GLeevec
it is a 2-phenylaminopyrimidine derivative
100
what protein kinsases does imatinib inhibit?
cellular ABL
viral ABL
p210 BCR-ABL
p185 BCR-ABL
TEL-ABL
etc
It inhibits only two other tyrosine kinases: the platelet-derived-growth-factor receptor and the stem-cell-factor receptor, c-Kit.
101
what does IC50 indicate?
Half-maximal inhibitory concentration (IC50) is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research.
the smaller the better
102
where does imatinib bind to inhibit BCR-ABL?
STI-571 targets the conserved nucleotide-binding pocket of Abl, with high specificity. (ATP cleft)
Imatinib is an inactive state inhibitor (Type 2)
103
what results were shown in the phase 3 clinical trials for imatinib?
Imatinib vs interferon-a/ARA-C - the responses of the two were compared to show the significance compared to conventional therapy at the time
CHR complete hematological response
CCR: complete cytogenetic response
both of these factor were measured and imatinib showed a signifcantly better percentage of activation compared to conventionel thereapy IFN
patient survival rate was also measured and after 60 months imatinib % was much higher at 95% compared to 89% IFN
104
why do we need to develop other inhibitors for BCR-ABL?
because with any therapy like imatinib we are putting a pool of cells under evolutionary pressure and so forcing escape mutants to form
other inhibitors are needed to target by other mechanisms so we can still inhibit the cells once they have mutated
105
what is the difference between the type 1 TKI PD166326 that was develop compared to the already established imatinib?
this one is an active state inhibitor (compared to type2 inactive state inhibitor than imatinib is)
the IC50 is better(IC50=8nM compared to IC50100-350nM)
however it does have some effects on the SRC family which could be dangerous (bcos kinases are more similar in the active state)
106
what are the 4 main resistance mechanisms to imatinib?
| clusters of mutants are in what regions?
Atp binding region – makes sense because this is what is involved with interaction with the inhibitor
T315 – forms a hydrogen bond with imatinib – so mutant no longer does (reducing affinity to imatinib)
SH2 interaction site M315
Activation loop aa379-398 - Activation loop mutants change the substrate availability (doesn’t affect interaction with the inhibitor)
107
the clinically approved second generation TKIs target what escape mechanism of BCR-ABL?
Efflux pumps – abl seems to escape evolution pressure by activating efflux pumps, cells can pump ot the molecules that they don’t want out of the cell
this can be imatinib for example
108
genetic screening of STI571 resistance mutants showed what?
you can over come the inhibition by mutating a wide range of site away from the bidning site
a good point of reflection for precision medicine – the more closely you interfere and work you may get escape mutants that you cant control by the same process, so you need other routes to deal with this.
109
give four example of 2nd geneation TKIs for BCR-ABL
* nilotinib - DFG out inhibitor
* dasatinib - DFG in inhibitor
* bosutinib - against non ABL dependent resistance mechanims (efflux pumps)
* ponatinib - SRC/ABL designed for T315 mutation. lower ic50 for almost all mutations
110
The modern “molecular medicine” based approach to cancer therapy requires ...
a) a deep understanding of the mechanism that is targeted
b) molecular structure informs the development of therapies
c) resistance to therapy can have multiple causes and may be therapy induced => Treatment is a dynamic process that is guided by better understanding leading to better diagnostic and more sophisticated intervention
111
how does localisation of ABL affect cellular fate
* ABL activity can lead to cell survival in response to growth factors concomitant with cytosolic localisation
* but also to cell death in response to DNA damage and oxidative stress concomitant with nuclear accumulation
## Footnote
its activity is context dependent
112
what are the two major mechanisms BCR-ABL has been implicated in malignant transformation
Two major mechanisms have been implicated in the malignant transformation by BCR-ABL1:
a) altered adhesion to bone marrow stroma cells and extracellular matrix, and
b) constitutively active mitogenic signaling and reduced apoptosis
