Kinases and Cancer

Question 1

what are the differences between conventional and modern cancer treatments

Answer 1

conventional - targets generic properties such as rapid proliferation
modern - targeting specific proteins/receptors - such as monoclonal antibodies

Question 2

what occurs from paracrine ErbB stimulation

Answer 2

ERB ligands are released from stromal cells

Question 3

what occurs from autokrin ErbB stimulation

Answer 3

activates metalloproteinases - cleaves pro-ERBB ligands

Question 4

what is the basis and idea behind EGF/ErbB receptor and kinase receptor intervention

Answer 4

ErbB receptors are dysregulated in wide range of cancers
1 - inhibit dimerisation of EGF/ErbB receptors to suppress proliferation
2 - inhibit kinase activity via small molecule tyrosine kinase domain inhibitors

Question 5

what is the mechanism of trastuzumab (herceptin) on Erb2

Answer 5

binds to the juxtamembrane region of Erb2 - inhibits proteolytic cleavage of Erb2 ectodomain
this is antibody-dependent cellular cytotoxicity

Question 6

what is the mechanism of action of pertuzumab on Erb2

Answer 6

binds directly to the Erb2 dimerisation arm
blocks both dimerisation and activation

Question 7

what is the mechanism of action of cetuximab (erbitux) on EGFR

Answer 7

competes with ligand for binding to EGFR
uses a dual mechanism:
- blocks the binding site on EGFR domain III
- steric inhibition of active EGFR conformaiton

Question 8

what is the mechanism of matuzumab on EGFR

Answer 8

does not compete for binding but reduces affinity of EGF for EGFR - in matuzumab presence EGF can only contact EGFR domain III - reducing affinity
interferes with formation of active EGFR

Question 9

what is the overall structure of kinases

Answer 9

N lobe and C lobe - hinge region in between
ATP binding site is located in the cleft between the lobes
N- lobe - 5 beta-pleated sheets and 1 alpha-helix
C lobe - mainly alpha helical

Question 10

what are the residues important for catalysis in kinases

Answer 10

present on both lobes
N lobe:
P-loop (glycine rich)
beta-strand 3
alpha-helix C
C lobe:
beta-strands 6/7
A-loop (activation loop)

Question 11

what is difference between an activated/inactive A-loop

Answer 11

inactive - tyrosine residues are unphosphorylated
active - tyrosine residues are phosphorylated
A-loop acts as a pseudo substrate

Question 12

what does chronic myelogenous leukemias (CML) effect

Answer 12

early haematopoetic stem cells

Question 13

what are the two phases of CML

Answer 13

acute - significant increase in immature white blood cells
chronic - elevated number of mature white blood cells

Question 14

what causes CML

Answer 14

reciprocal translocation between chromosome 9 and 22

Question 15

what are the key regulatory domains for Abl regulation and their functions

Answer 15

SH3 - poly-proline binding domain
SH2 - phosphotyrosine recognition domain

Question 16

what are some of the features of Abl

Answer 16

Abl knockout in mice is lethal
ABL shuttles between the nucleus and the cytosol - in response to ECM adhesion

Question 17

features of the basal state of ABL and what maintains it

Answer 17

basal state - low phosphorylation activity, in an off state
maintained by:
cis - self interactions
trans - interactions with range of targets

Question 18

what are the essential features of maintaining the auto inhibited state of ABL

Answer 18

association of SH3 with its N-lobe
insertion of myristol group in the C-lobe

Question 19

outline partial activation of ABL

Answer 19

1 - unlatching - removal of myristyl group from C-lobe leads to partial activation
2 - unclamping - displacement of SH3 from N-lobe

Question 20

how is full activation of ABL achieved

Answer 20

switching - phosphorylation of tyr-412 on the activation loop and tyr-245 in the SH2 kinase linker

Question 21

how is cis/trans activation of ABL regulated

Answer 21

inhibitor and activator proteins

Question 22

how does ABL lead to cell survival

Answer 22

in response to growth factors concomitant with cytosolic localisation

Question 23

how can ABL lead to cell death

Answer 23

in response to DNA damage and oxidation concomitant with nuclear accumulation

Question 24

what odes the lack of SH3 domain of GAG-ABL indicate

Answer 24

suggests that the lack of SH3 - N lobe interaction leads to constitutive activity

Question 25

what does the lack of a myristate group in the BCR-ABL group suggest

Answer 25

suggests that lack of myristate group insertion in C lobe leads to constitutive activity

Question 26

function of the BCR domain

Answer 26

mediates dimerisation and association of effector proteins

Question 27

what are the main problems for targeting ATP to inhibit tyrosine kinase phosphorylation

Answer 27

specificity problem - 512 kinases and ABL and BCR-ABL share the same ATP binding site
abundancy - ATP is present at 2-5nM so need a very high affinity substrate to compete

Question 28

what is able to inhibit the ATP phosphorylation of Abl

Answer 28

Imatinib - inhibits it 50%

Question 29

how odes imatinib inhibit ABL kinase domain

Answer 29

targets the conserved nucleotide-binding pocket of ABL with high specificity

Question 30

what other tyrosine kinases does imatinib inhibit

Answer 30

platelet derived growth factor receptor
stem cell factor receptor

Question 31

what type of inhibitor is imatinib

Answer 31

it is an inactive state inhibitor (type 2)
exposes the allosteric pocket by removing DFG

Question 32

what inhibitors does ABL 1 complex with

Answer 32

type 2 imatinib mesylate
type 1 TKI (active state inhibitor)

Question 33

what are the 2nd generation TKI’s and their specific purpose

Answer 33

nilotinib/bosutinib - against non-ABL dependent resistance (efflux pumps)
dasatinib/ponatinib - SRC-ABL T351 mutations, lower IC50s for almost all mutations