Module 4- Molecular Architecture Of Signalling Switches

Question 1

Diversity of RTK mechanisms

Answer 1

Most RTK turned on by dimerisation, the way that this dimerisation can happen is diverse and differs with different RTKs

Question 2

EGFR dimerisation

Answer 2

Single ligand binds to single receptor within the large extracellular domain of the receptor
This triggers a large conformational extracellular change leading to dimerisation through a different interface
Dimerisation allows kinase activity

Question 3

Different EGF family receptors and their features

Answer 3

EGFR- various ligands binding to large extracellular domain with an active intracellular kinase
HER2- extracellular domain has no known ligands, constitutively active looking for other R to dimerise with, active intracellular kinase
HER3- large extracellular domain binding to NRG1/2, inactive intracellular kinase
ErbB4- active extracellular kinase sensitive to different ligands than EGFR and active intracellular kinase

Question 4

EGF family receptor dimerisation consequences and mechanism

Answer 4

Large conformational change releases auto-inhibition of receptor stalk through breaking the beta-hairpin
Opens the possibility for dimerisation through the beta-hairpin
Dimerisation through the beta-hairpin is conserved between different family members so heterodimers can form

Question 5

ErbB2 (HER2) orphan receptor features

Answer 5

No known ligands
Amplified in cancers (breast ~15-30%, endometrial and uterine)
Often amplified in conjugation with Grb7 SH2 adapter
Doesnt have autoinhibition arm or require a ligand for activation
Can form heterodimers freely
Preferentially forms heterodimers with Her3

Question 6

Glycosylation of receptors

Answer 6

Important for extracellular parts of proteins
N-linked and required for proper processing/ trafficking of receptor
Can influence ligand binding and antibody sensitivity

Question 7

Activation of the kinase domain

Answer 7

Alpha-c helix, DFG and activation loop must be aligned correctly for activation
There are multiple ways kinases can be off- ac helix out of alignment, activation loop disordered or both not correct
The way a kinase is activated depends on its function in the pathway

Question 8

Activation of a kinase allosterically from its partner- how was this discovered

Answer 8

One kinase is active, one is inactive
Inactive kinase allosterically activates the receiver/ activated kinase
Conformation is important for kinase activity
Looked at level of EGFR phosphorylation through mutations: found that one kinase must be active and they must be in the right arrangement for kinase activity to occur

Question 9

Kinase activity and mutation in cancer

Answer 9

Kinase activating mutations frequently occur in breast and other cancers
Mutations often destabilise inhibited conformation at the active site
Kinases have large druggable active sites and can potentially target mutated kinases specifically

Question 10

Anti-Erb therapeutics

Answer 10

Extracellular domains: humanised monoclonal antibodies that bind to the extracellular region
Intracellular domains: small molecule inhibitors of kinase domain that bind ATP site

Question 11

Three examples of therapeutic monoclonal antibodies to block Erb dimerisation

Answer 11

Cetuximan- blocks EGFR binding to L= EGFR specific
Traztuzumab (Herceptin)- blocks Her2 by binding to stem of the receptor= Her2 specific- effective against Her2 homodimers, heterodimers can still form
Pertuzumab- blocks dimerisation arm of Her2 directly, blocking all dimerisation= Her2 specific

Question 12

Clinical mutations and therapeutic efficacy

Answer 12

Mutations may affect the receptor dimerisation or monoclonal antibody binding
Mutations can make a receptor more or less susceptible to therapies

Question 13

Overall features of the Ras superfamily

Answer 13

5 branches of family, each with different members, all sorts of signalling roles
All have common core, different regulators
Core good for controlling other proteins and for being controlled by other proteins
Ras family involved in general signalling, growth/ survival and migration= related to cancer so mutation is common

Question 14

Key features of Ras control

Answer 14

Terrible enzyme (GTPase) but good switch- bind GDP/GTP very tightly- pM affinity
Not good at controlling its own nucleotide exchange, controlled with GEF/GAP
Need GTP exchange factor (GEF) to get GTP in the active site for activation
GTPase reaction to turn off very slow, need GTPase activating protein (GAP) to inactivate
Controls many different things when in active GTP bound conformation

Question 15

Anatomy of the Ras switch

Answer 15

Two switches involved
When GTP is bound, hydrogen bonds can be formed with these switches and the third phosphate= conformational change and activated, meaning effector complexes can form and downstream activation occurs
When GDP bound, H bonds cant form as the third P is missing, inactive state and effectors bind weakly so Ras cannot bind or activate other proteins
From inactive to active, binding affinity for Ras and its effects increases ~1000 fold from microM to nM

Question 16

The three Ras siblings0 Harvey, Kirsten and Neuroblastoma

Answer 16

Three isoforms with a common structure
Conserved N-terminus, with divergent C-terminus. All C-terminus have CAAX box which is a motif for lipid tail PTM so that it can be inserted into the membrane
Different mutations are common in each which lead to cancer- prototypical patterns in different cancers

Question 17

How does GAP inactivate Ras and how this relates to cancer

Answer 17

Transition state mimic complex is formed
Arginine finger binds to the P in GTP= interacts with the transition state complex and needs to be in the correct place- GAP glutamine 61 (Q61) forms interaction with this Arg and the P leaving group so stabilises the interaction and transition state- mutating this in cancer breaks the switch
Any side chain mutation of glycine 12 creates steric hinderance and blocks the P leaving so switch is broken and stays on

Question 18

How do GEFs turn on Ras

Answer 18

Eg SOS- sticks a helix into the GTPase active site and displaces the GDP nucleotide inside
When SOS leaves, Ras binds whatever nucleotide is there
[GTP] is about 10x greater than [GDP] in the cell so it is most common that it then binds GTP and become activated

Question 19

Measuring exchange factor activity with fluorescent nucleotide analogues

Answer 19

Load the cell with MANT-GDP which fluorescently labels all GDP
Remove the excess GDP, leaving only GDP bound to Ras
MANT-fluorescence increases when bound to GTpase
When GDP is kicked out of the active site there is low fluorescence
Measures the activation of Ras

Question 20

Membrane environment and Ras activation by SOS
Ras density on membrane and rate of release

Answer 20

Looking at with MANT fluorescence
When you add free ras without PTM to be in membrane and catalytic part of SOS, GDP is released at a slow rate
When Ras is in the membrane the catalytic rate of activation by SOS increases= membrane environment is important for enhancing
Increased density of Ras causes increased rate of activation= feed forward activation

Question 21

Why is Ras density in the membrane important

Answer 21

SOS has membrane binding domains
In the off/ autoinhibited state these membrane binding domains are non-functional
When opened up and active, the membrane binding domains are active and allosteric Ras can come and bind at the rear which helps the exchange of nucleotides to occur in substrate Ras as feed-forward

Question 22

Overview of Ras activation by SOS

Answer 22

phosphate recruits Grb adapter that binds to receptor P and also binds to SOS- bringing SOS to the membrane
SOS then activates little Ras GDP->GTP, and then membrane parts of SOS can bind to the membrane
Allosteric Ras then binds and further activates SOS= amplification
Regulation by membrane and by proteins

Question 23

Mutations in SOS and Noonan syndrome

Answer 23

1 in 1000-2500 births
~85% have congenital heart defect
Other symptoms= short stature, learning difficulties, characteristic appearance
RASopathy (thing which disrupts Ras signalling)- mutations often disrupt autoinhibition= more activation of Ras
Variants binds and activate Ras more readily than the wildtype 9seen in western blot)

Question 24

Pros and cons of monoclonal antibody therapies

Answer 24

Pros: highly specific, good distribution and long half life
Cons: expensive to develop and produce (COST!), immunogenecity, high specificity can be con sometimes

Question 25

Pros and cons of small molecule kinase inhibitors

Answer 25

Pros: cheaper development, cheaper production, immunogenecity not a problem, can access intracellular and distribute freely Cons: more off target potential and potential short half life

Question 26

Arms race with cancers and resistance to EGFR blockade therapies

Answer 26

Mutations occur in downstream parts of pathway- kinases and GTPases to make the pathway constitutively active Resistance develops against the therapies being treated with Looking for drugs against the downstream molecules which have been mutated to be constitutively active

Question 27

Why kinase inhibitors have been developed so easily and rapidly

Answer 27

ATP binding to kinases is weak in microM-mM range Also have large active site with lots of chemical space to operate and can generate small molecule inhibitors at high affinity to bind and inhibit the active site region

Question 28

Why drugs against Ras have been so difficult

Answer 28

GTP/GDP binds at a very tight affinity at pM and there is not much free space for the binding of small molecules in the active site Small relatively rigid active site- not much room Need to be tighter binding than GTP/GDP pM affinity and need to have space to fit in

Question 29

Kinase inhibitor development being narrow? Specificity?

Answer 29

Limited number of targets have been explored and there are more potential targets to come Not very specific- can have off target toxicities and activities in other kinases- can take advantage of this so pro and con

Question 30

Possible mechanisms of modulating Ras activity

Answer 30

Change localisation of Ras- block it from getting to the membrane to dampen the signal Block the on switch (GEF) Common mutations in particular isoforms and particular cancers being taken advantage of Synthetic lethality- things they cooperate with through an indirect approach

Question 31

Changing Ras localisation as a potential therapy

Answer 31

Farensyl transferase catalyses the first step in the PTM pathway to generate the lipid tail for insertion into the membrane Inhibitors of this were in phase III clinical trials but failed as there is an alternative prenylation path in K and N Ras driven cancers that causes the lipidation so signalling can still occur Showed promise but not really effective

Question 32

Decreasing levels of active Ras by interfering with GEF on switch

Answer 32

Identified a binding pocket abutting switch region Used fragment based screening approach and NMR Crystal structure show similar binding, and weak affinity Weak affinity suggests the approach was difficult

Question 33

Looking at mutant-specific Ras inhibitors

Answer 33

Takes advantage of certain mutations in Ras isotyoes In K-Ras G12 is commonly mutated- in nearly 50% of lung ardenocarnimonas it is G12C Cystiene is highly nucleophilic so potential to attack with inhibitor Was specific- doesnt touch WT and causes covalent modification in active site which allows it to compete with the pM binding affinity of GDP/GTP- bound in position near the third P binding site Effectively blocks SOS activation and has cell line specificity for G12C

Question 34

Looking at synthetic lethal partners of Ras to try and target it

Answer 34

Cancers which have mutated Ras to be constitutively active then rely on its pathway for driving its growth There are many pathways Ras is involved in- aim to look at different areas and effectors in these pathways to exploit to prevent the growth from this pathway

Question 35

The steps in ubiquitination (ubiquitin conjugation)

Answer 35

E1 ub activating enzyme forms transient covalent bond with ub and uses ATP to activate it Conjugating E2 enzyme takes ub from E1 making a relatively liable thioester bond with a cystiene residue in E2 active site E3 brings substrate and charged ub together and provides specificity, forming an isopeptide bond with a lysine residue Is a reversible cycle (by de-ubiquitinases) and fate of the ub-protein depends on how the chain is built

Question 36

Features of ubiquitin

Answer 36

C-terminus needs to be activated, attaches to other proteins here N-terminus has nitrogen 76 residues with 7 different lysines- all with nitrogens

Question 37

Ubiquitin modifications and the different outcomes

Answer 37

K48 and K11 chains result in proteasomal degradation K63 linear chains result in scaffolding functions Mono-ubiquitination eg on histone proteins to control chromatin compaction

Question 38

Different numbers of each ubiquitination enzyme- link to specificity

Answer 38

2 types of E1 ligases- not much specificity from this ~50 E2 ligase types which can influence chain type Hundreds of different E3 ligases (~1000) which select different substrates and provide specificity for the fate of the substrate

Question 39

Cullin E3 ligase

Answer 39

Molecular machine 8 different cullin proteins, with multiple different adapters and receptors Allows diverse degradation complexes using the same scaffold

Question 40

Opportunities for drug development in the ubiquitin cascade

Answer 40

Specificity increases across the pathway and with E3 ligases could make specific drugs to control specific substrates Inhibiting proteasome function- less specific De-ubiquitination could be inhibited to stabilise some substrate-ub complexes

Question 41

Proteasome inhibitors and example

Answer 41

Have 26S lid which recruits and senses ub chains on substrates Internal 20S proteasome barrel with multiple protease activities where cleavage occurs- where inhibitors are made eg MG132 MG132 (bortezomib) found to cause decrease in cell growth and more ubiquitin chains are seen Bortezomib approved for treatment of multiple myeloma- leads to high tumour suppressors eg p27 and p53 and induces cell death. Not overly specific, there is a window of opportunity for effectiveness as cancer cells are more reliant on the proteasome than normal cells due to their high turnover rate

Question 42

Targeting E3 ligases for specific drugs

Answer 42

There are hundreds of E3 ligases which are specific for different things. Could increase or decrease the degradation of a specific substrate Eg tumour suppressor decreased/ downregulated or oncogene increased/ upregulated Eg MDM2 inhibitor which is the known p53 E3 ligase prevents it from being ubiquitinated and degraded

Question 43

How does thalidomide work and the issue with it

Answer 43

Binds cereblon cullin E3 substrate adapter protein, creates neo-substrates/ new substrates that wouldnt normally bind and wouldnt normally be degraded For example, TFs (IKZF family) important in early development of limbs were degraded inappropriately, leading to baby deformalities and 50% fatality

Question 44

How did they find that thalidomine was toxic/ what was the cause of the toxicity

Answer 44

In rat and mouse animal trials, there is a single residue difference in the scaffold adapter binding pocket so didnt produce the same toxic effects due to a difference in thalidomine binding

Question 45

Anticancer agent from thalidomine failure

Answer 45

IKZF TFs are important in B cell lymphomas and multiple myelomas as they suppress IL-2 expression so therefore could be anti-cancerous Showed there was a decrease in levels of IKZFs with increasing thalidomine concentrations due to increase in IL-2

Question 46

PROTACs from thalidomine failure

Answer 46

Proteolysis targeting chimeras Fuse together an E3 targeting component (eg cereblon or VHL) with a linker to a substrate specific recruiting protein (warhead that can be switched out) Enables recruitment of new substrates to E3, causing ubiquitination and degradation of substrates which arent normally targets of the system

Question 47

Advantages of targeted degradation over inhibition

Answer 47

Inhibition can be overcome with resistance, and in kinases only one within the dimer needs to be active for function to occur Sustained pathway inhibition from degradation Is catalytic rather than relying on blocking, so no 1:1 stiochiometric ratio is needed Scaffolding roles are eliminated