Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOL3017 - Molecular and structural basis for disease > Cancer kinases > Flashcards

Cancer kinases Flashcards

1
Q

Conventional s modern cancer therapy

A

‘conventional’ = targeting generic properties e.g. proliferation, using cytotoxic agents/chemo

‘modern’ = Targeting specific properties of C cells e.g. receptors or proteins that are dyregulated - uses mAbs, Kinase inhibators

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Explain proteins controlling cell growth/proliferation

A

Signalling molecule
Signalling receptor - often a protein kinase
intracellular transducers
Secondary emssengers (phosphorylated proteins
Transcription factors
Apoptotic proteins, cell cycle control proteins, DNA-repair protiens made

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Genetic bases of cancer

A
  1. Gain of function mutations - conver proto-oncegenes to oncogenes
    e.g. Point mutations, Chromosomal translocation, aplification
  2. Tumour supressor genes - stop working
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What Kinases are on the ‘mutated list’ for cancer

A

*receptor tyrosine kinases (RTK): e.g. EGF/ERB

*cytosolic kinases:e.g. Abl, src

*Nuclear kinases: e.g. Jun Fos

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Why is there a specificity prolem with kinases

A

512 kinases in human genome, targeting a particular one = difficult

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Two kinase dysregulations that have been successfully targeted in modern cancer therapy

A
  1. EGF/ERB family of receptor tyrosine kinases (RTK): EGF/ERB
  2. cytosolic kinases: Abl
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Paracrine ErbB stimulation

A

ERB ligands released from stromal cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Autocrine ErbB stimulation

A

ER/Src, GPCR or FZD stimulation activates metalloproteinase cleaving Pro-ERBB ligands

metalloproteases becoming another target for cancer treatment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Normal EGFR/ErbB signalling

A

Ligand induced dimerisation on the outside triggers increased 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 e.g. MAPK, PI3K-AKT (to mTOR), Src, STAT

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Why is there not abberant sgnalling of EGFR?

A

Because EGF isnt always present

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Explain the basis and idea for intervention of ErbB

A

ErbB receptors are aberrantly regulated in a wide range of cancers

  1. Inhibit dimerisation of EGF/ERbB receptors to suppress proliferation signal
  2. Inhibit kinase activity using small molecule tyrosine kinase inhibitors

example of molecular medicine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Different names for the ErbB receptor family

A

EGFR = ErbB1
Her2 = ErbB2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

4 members of the ErbB family, which one to target?

A

NB: class of ligands, family of receptors

Haynes 2005

multiple permutations of the receptors are possible:

ERB2 interacts with all 4

But not all possible e.g. ERB4 doesn’t interact with ERB1

potentally interesting to look at: 2/3 receptor combination as ERB2 -> MAPK and ERB3 -> PI3K-AKT

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Explain structure of the ectodomain

A

Crystal structure of EGFR (but whole family share similar structure) have been discovered (Burgess 2003) - both ligand free and ligand bound state

4 domains - I, II, III, IV

without ligand an interface between domain 2 and 4 is made - in this state dimerisation isn’t possible

When EGF comes, it binds between domains 1 and 3, which are not close to each other, the joining if these domains by EGF causes an extended version if the ectodomain to form. Domain 2 is now exposed with a beta strand protrusion forming a dimerisation site with another activated EGFR receptor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

From the known structural formation of the EGFR/ErbB dimers, what therapeutics could me made to stop this

A

EGFR, ErbB3 and ErB4 ectodomain undergo ligand induced reorganisation making receptors dimerisation competent

meaning that there could be control/targeting here - either a competative inhibator (e.g. mimics EGF) that keeps the inacive conformation intact, or a molecule that blocks dimerization (e.g. by binding to the beta strand protrusion of domain2)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Explain how ErbB2 is different from theother family members in structure

A

(Burgess 2003)1,3, and 4 all have an inactive folded state, whereas ErbB2 in its ligand free state looks similar to EGFR when its bound to EGF - its already dimerisation competent, this is what makes it prone to act with the other receptors of the family (this is also why theres no ERB2 homodimer)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Since we cannot interfere with ErbB2’s ligand binding, what can we still interact with?

A

Can still block dimerisation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Working model for ErbB3/2 homo and hetero dimerisation

A

ErbB3 requires ligand (e.g. NRG) to get into an upright dimerisation competent conformation
can then interact with ErbB2 , allowing cross phosphorylation and activation of signalling

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How to target receptor ectodomains

A

Carter 2001
Antibodies - use mouse antibodies (but can lead to cross-reaction)

Chimeric antibodies - use human form but with Variable regions of the mouse antibody

Humanized antobodies - keep human antibodies but keep some of the recognising sequences from mouse antibody

Optimal - go for human antibodies entirely, but still needs to be presicly tailored

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How can we generate antibodies with the right characteristics/what are these characteristics?

A
  • recognise target
  • Target for destruction either by Complement binding or Fc receptor binding (complement dependant cytotoxicity or antibody-dependant cellular cytotoxicity)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Current/in clinic EGFR/ErbB targeted therapies

A

Theres a whole class of antibodies, against different targets, that are currently in the clinic e.g.
- Herceptin (humanized mAb) - targets ErbB2 - breast cancer treatment
- Omnitarg (Humanized mAb)- Phase II clinical trials, also targets ErbB2
Erbitux (Chimeric mAb) - targets EGFR - approved for treatment of colorectal cancer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Mechanism of Antibodies against ErbB2

A

Leahy 2008
Trastuzumab (Herceptin):
- Binds to juxtamembrane region of Erb2
- Blocks proteolytic cleavage of Erb2 ectodomain
- avoids remaining active kinase
- anti-body dependant cellular cytotoxicity

Pertzumab:
- binds directly to both the Erb2 dimerisation arm and blocks both dimerisation and activation (binds to domain 2)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

One downside of Herceptin (Trastuzumab)

A

Primary or acquired resistance

may be able to overcome this by identifying biomarkers for its effects in patients, and then only administering it to patients that are thought to respond

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Mechanisms of antibodies against EGFR

A

Have the addition of being able to effect the activation step as well as dimerization

Cetuximab (Erbitux) - competes w/ ligand for binding to EGFR using a dual mechanism:
a) Blocks the ligand binding site on EGFR domain III
b) Steric inhibition of extended, active like EGFDR conformation

Matuzumab - binds to domain III of EGFRs non-overlapping site of EGF
- doesn’t completely compete for EGF binding ti EGFR
-Does reduce apparent affinity of EGF for EGFR
- Interferes with formation of active-like EGFR
- EGF is only able to contact domain I or II, not both, therefore reducing its affinity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Why is there clinical benefit for Cetuximab and martuzmab to be used in combination therapy

A

Dont compete for binding

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What have preclinical studies shown is a limitation to trastuzumab

A

ErBB ligands can circumvent Tmabs ability to block downstream signalling and proliferation (likely due to signalling competent heterodimers still being able to form)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Kinase function

A

use the gamma phosphate of ATP and put it onto a residue
1. autophosphorylation - residue on kinase itself
2. Target molecule - residue on target protein (requires Mg2+)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Overall structure of kinases/kinase domain

A

Two lobes connected by a hinge region:

N-lobe - 5 stranded beta sheets and a single helix

Larger C lobe - mainly a-helical

ATP binding site located in cleft between the lobes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Residues of kinases important for catalysis:

A

From both lobes

N lobe: P loop/nucleotide binding loop (Gly rich)
Helix C - good handle visually to see active vs inactive

C lobe: Catalytic loop, activation (A) loop - contains a DFG motif - around 20 residues

30
Q

What kinase acts as a template for how kinases work

A

Insulin receptor kinase (IRK)

31
Q

Structure of IRK in active and inactive conformations

A

Inactive state: Conformation of activation loop very different - acts as a pseudosubstrate, binds onto the site where a substrate would usually bind, Helix C also has a different angle and the DFG motif is turned outwards

Active state: A loop protrudes away from the molecule, tyrosine residues have phosphates attached

32
Q

Kinase autoinhibition (based off of IRK)

A

A loop acts as a pseudosubstrate

phosphorylation of Tyr/Ser/Thr residues in A loop causes significant rearrangment of the A loop allowing subsequent substrate phosphorylation

33
Q

Due to kinase studies being done primeraly in IRK, what was a surprise when the structure of EGFR kinase was discovered (Burgess 2003)

A

Activation loop ofd EGFRK was similar to the active conformation of IRK (A loop facing out), so autoinhibition does not control EGFRK in the same way, its already in its active form

34
Q

If EGFRK doesn’t posses the autoinhibatory activation loop conformation that IRK does, how is it kept inactive

A

Controlled through interactions with the N-terminal domain

35
Q

Recent observation of asymmetric EGFK dimer

A

EGFR kinase is controlled by self interactions with another EGFRK

Dimer is asymmetric

C-lobe interaction of the donor (1) kinase with N lobe of acceptor (2), kinase activates acceptor (2) but not donor (1) kinase

Inhibition by N-lobe is seen in many kinases e.g. Src (SH2/3)

36
Q

Model for EGFR ectodomains and kinase association put together

A

Inactive EGFRK, Ligand comes about (EGF), causes receptor to form upright conformation, allowing self interaction with another receptor, asymmetric dimerisation of EGFRK, causing trans-phosphorylation from the donor kinase to the Active acceptor kinase

37
Q

ATP binding site of kinase

A

ATP site between the N and C lobes
Broadly, 2 interaction sites:
Hydrophobic pocket 1 on C lobe
Hydrophobic pocket 2 on N lobe

also ability to affect allosteric site - reason is due to conformational change between inactive and active state

38
Q

Reasons that ATP is less targeted for effecting kinase functuon, and if it was sussessfull

A

Highly abundant in cells, so would need a massively high concentration of competitive inhibitor

ATP is used in lots of cellular processes by lots of enzymes and proteins, it is not a good specific target (therefor off target effects)

Despite this - Molecules exploiting ATP interaction sites for inhibitors were created, there were various chemical scaffolds used, there have been expanded into kinase inhibitors that have nm EC50s and kds that can be used as drugs!

39
Q

2 types of kinase inhibators

A

Type 1 and type 2, target the inactive and active state

40
Q

EGFR kinase inhibitor complexes

A

AMP-PNP - ATP like molecule that cannot be hydrolysed (NOT USEFULAS A DRUG JUST FOR INTERACTION STUDIES)

Iressa - intecats with site I and II - DFG-in, active kinase
Lapatinib - binds to allosteric site (only available in inactive state) - DFG out, inactive kinase

41
Q

How were Irreversible inhibitors of kinases created

A

Using reactive kinase cystine residues - can make compounds that exploit this by forming covalent bonds with the cystines e.g. HKI-272 (clinical trials?)

42
Q

Irissa

A

TKI (tyrosine kinase inhibator)
used for treatment of NSCLC

43
Q

Why is it important to create inhibators for multiple stages of the kinase cascade of cell proliferation?

A

Escape mutants are occurring in trials, within 6 months a treatment may stop working as cancer cells have found a way round it, important to try target at as many points as possible with as many different mechanisms as possible e.g. (Kinase active and inactive state)

44
Q

Chronic myelogenous leukemia (CML)

A

Accounts for ca 15% of all leukemias

Affects early hemapoietic stem cells

Elevated numbers of mainly mature white blood cells

45
Q

Disease progression of Chronic Myelogenous Leukemia (CML)

A

Chronic phase (may last months to years)

  • Elevated numbers of (largely mature) while blood cells

Acute phase
* Significantly increased number of largely immature white blood cells

46
Q

What is philidelphia chromosome

A

In CML, patients chromosome 22 extreamly short

  • suggests a causal relationship between chromosome abnormality and chronic granulocyte leukemia (Novell 1960)

reciprocal translocation between chromosome 9 and 22 - creating the BCR-ABL gene(Rowley 1973)

47
Q

How was BCR-ABL established as a target for leukemia

A

ABL oncoproteins were seen to transform primary cells and generate leukaemia in vivo - late 80’s (1989-90)

48
Q

Abl primary structure

A

N terminus: Myristate group (help proteins localise to cell membranes), Kinase domain, SH2 and SH3 (regulatory domains)

C terminus: localisation cues - DNA binding domain, NLS and -F-actin binding domain

49
Q

SH3

A

Poly proline binding domain: recognises PxxP motif

50
Q

SH2

A

Phosphotyrosine recognition domain

51
Q

Essential features for maintaining the auto inhibited state of Abl

A

association of SH3 with N-lobe

  • Insertion of Myristol group in C-lobe
52
Q

Route to partial activation of Abl

A

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 (autophosphorylation) in SH2- kinase linker

RW

53
Q

What makes the primary structure of BCR-Abl and vABL different from the normall Abl structure

A

BCR at N teminus instead of the myristate region

vABL - has a GAG protein to allow virus to get into cells – missing SH3 (sometimes also referred to as GAG-Abl)

54
Q

What does Abl activity lead to

A

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

55
Q

In terms of Abl, what does the cellular fate of the cell depend on

A

ABL localization

56
Q

Explain activation of ABL kinase

A

Goes from being trans inhibited, to still autoinhibited, to activation when a substrate activator comes along

57
Q

What is the master switch of the kinase

A

RW

58
Q

Explain the primary structure of BCR

A

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.
CaLB: putative Calcium dependent lipid binding domain with activation function of Rac-GTPase (RAC-GAP)

Arrows in diagram: breakpoints in BCR-ABL fusion leading to three fusion proteins. Most prevalent is P210 fusion withABL

Transfucion doesn’t mean youll get the full version of BCR-Abl – could have a shorter section – different versions

59
Q

Explain hoe BCR self-association enhances phosphorylation activity

A

RW

60
Q

What are the outcomes of BCR-ABL activation

A

Altered adhesion, mitogenic activity, inhibition of apoptosis - all lead to a malignant phenotype (transform to cancer cells)

61
Q

3D structures of GAG-ABL and BCR-ABL

A

3D-Structures of full length BCR-ABL and GAG-ABL are not available

62
Q

Explain what the differences in structure from normal Abl of GAG/BCR-ABL suggest

A

GAG-ABL (v-ABL) lacks SH3 domain – Suggest that lack of SH3-N-lobe interactions lead to constitutive activity

  • BCR ABL lacks myristate group – Suggest that lack of myristate insertion in C-lobe leads to constitutive activity
  • BCR domain mediates dimersation and association effector proteins
63
Q

Inhibition of BCR ABL

A

ATP required for tyrosine phosphorylation activity

  • Tyrosine Kinase inhibitor (TKI)
  • Alternatives: e.g substrate inhbators e.g. (Tyr) competitors - Early attempts did not yield successful inhibitors

RW

64
Q

Challenges for TKI inhibitors

A

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?

b) 512 Kinases in the human genome

c) Significant numbers of ATP dependent proteins ( enzymes) other than kinases –

ATP is abundant (2-5mM concentration in cells) so need high affinity competitor

65
Q

Inhibition profile of Imatnib

A

STI-571, also known as Gleevec or imatinib mesylate, is a 2-phenylaminopyrimidine derivative

Various forms of BCR-abl tested against P210 and P185 – same as in.. RW

66
Q

Imatinib ABL Kinase domain complex

A

STI-571 targets the conserved nucleotide-binding pocket of Abl, with high specificity. It inhibits only two other tyrosine kinases: the platelet-derived-growth-factor receptor and the stem-cell-factor receptor, c-Kit (Crystal structure shown by Ha ntschel 2004)

Imatinib is an inactive state inhibitor (Type 2 inhibitor)

67
Q

Explain phase 3 clinical trials of Imatnib

A

Higher response, lower intolerance in Imatinib compared to interferon-a/ARA-C (Druker 2008)

68
Q

Explain development of further BCR-ABL inhibators after

A

Abl1 complex with type 1 TKI (PD166326) (active state inhibitor)

Both against SRC and ABL

Significant improvement on Kd (compared to imatinib)– need less amound to bind (higher affinity) (Zang 2009)

69
Q

STI-571 (imitinab) resistance mutants

A

Main resistance mechanisms Cluster in four regions:

  • ATP binding region 248-255
  • T315 (H-bond with imatnib)
  • SH2 interaction site M315
  • Activation loop aa379-398
70
Q

Clinically approved 2nd generation TKIs

A

Nilotinib – DFG out inhibitor

Dasatinib – DFG in inhibitor

Bostutinib - Against non ABL dependent resistance mechanisms (opperates on eflux pumps)

Ponatinib - SRC/ABL designed for T315 mutations Lower IC50s for almost all mutations

71
Q

Genetic screen for STI-571 mutants

A

RW

BIOL3017 - Molecular and structural basis for disease (6 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions