Inherited mutations in CFTR and personalised cystic fibrosis therapy

Question 1

When was cystic fibrosis first described?

Answer 1

in 1930s

Question 2

What does the name cystic fibrosis refer to ?

Answer 2

refers to scarring and cysts in the pancreas

Question 3

How is cystic fibrosis often first detected?

Answer 3

by presence of salty skin/sweat due to increase in ionic content of sweat

Question 4

What tissues are affects in CF?

Answer 4

many tissues are affected and mainly secretory epithelia and exocrine glands

Question 5

What is the major problem with CF?

Answer 5

major problem with the lungs: regular infection, breathing difficulties- mainly only the lung infections are treated
- symptoms treated, not the underlying cause

Question 6

What type of disorder is CF?

Answer 6

it is a recessive genetic disorder

- it is a more prevalent type of recessive genetic disorder

Question 7

When was the CFTR gene discovered?

Answer 7

identified in 1980s

Question 8

How many caucasians carry a defective allele for CFTR and how many are affected?

Answer 8

1 in 25 caucasians have defective allele and 1 in 3000-4000 are affected

Question 9

What does the CFTR gene encode?

Answer 9

it encodes a membrane protein of the ABC transporter family - homologous to other members of ABC

• nucleotide gated chloride channel, ATP hydrolysed during activation cycles - during gating cycles
• activity is dependent upon phosphorylation by PKA

Question 10

How many different mutations have been identified in CF and what is a common mutation?

Answer 10

over 1000 different ones

- about 70% (90% in US) have the delta F508 mutation = phenylalanine deletion

Question 11

What does the CFTR channel look like ?

Answer 11

made up of a single peptide with 2 homologous domains

• predominantly phosphorylated following activity of protein kinase A
• phosphorylation of the R domain causes enhanced activity of the anion pore

Question 12

What are normal airways like ?

Answer 12

requires a balance between sodium and chloride ions to get correct height of pericilliary liquid
- height/volume of pericilliary liquid between the surface of epithelial cells
has to be a certain height to allow cilia to beat to remove mucus
height is determined by homeostasis of water and sodium and chloride ions

Question 13

What are the airways like in betaENaC transgenic mice/CF?

Answer 13

Transgenic mice have an overly active ENaC to induce symptoms
in CF it is caused by a decrease in CL- channel activity which alters the balance
cilia cant move as well
GAGs, chemokines and growth factors are not gotten rid of which causes infections
CF patients have a loss of sodium and chloride balance due to a lack of cl efflux due to loss of function mutations in CFTR

Question 14

What are they trying to do to improve treatments for CF?

Answer 14

try to develop taylor-made treatments for each individual based upon their mutation

Question 15

What are class 1 mutations ?

Answer 15

CFTR is not fully synthesised (e/g nonsense)

• due to nucleus function
• produces a truncated protein because the mRNA produced contains a nonsense mutation

Question 16

What are class 2 mutations ?

Answer 16

CFTR is retained in the ER (e.g. deltaf508)

- ends up being degraded by ERAD

Question 17

What are class 3,4,5 and 6 mutations?

Answer 17

present in plasma membrane but dont function properly

• class 3: impaired CFTR activation
• class 4: reduced chloride conductance- reduced open probability- may only be open for a very short period of time
• class 5: CFTR synthesis partly defective- fewer channels at the plasma membrane
• class 6: altered ion permeation - very rarely occurs , opens normally but few ions flow through per cycle

Question 18

What is important about the inheritance of alleles in CF?

Answer 18

inheritance of any 2 mutant alleles causes CF-dont have to be the same alleles they can be 2 different types of mutations
e.g deltaF508/deltaF508 or deltaF508/G551D

Question 19

What are the therapeutic strategies for CF?

Answer 19

ANTIBIOTICS- mainly just focusing on treating symptoms - treating infections that occur due to improper removal of substances from the lungs
GENE THERPAY- starting to try and treat the underlying causes of the disorder- idea of delivering a working copy of the gene to the lungs

Question 20

Why is it difficult to deliver a working copy of the gene to the lungs?

Answer 20

due to the thick layer of mucus in the CF it is difficult to administer exogeneous substance

Question 21

What is pharmacotherapy treatment ?

Answer 21

it is when the function of the mutant CFTR is tried to be restored
- targetting a specific type of mutation and try to make it work more like WT

Question 22

What are the altered signialling pathway that could be targeted in CF treatment?

Answer 22

try to increase cAMP

- cAMP can stimulate ion channels so increasing it should increase activity of channels via phosphorylation of PKA

Question 23

Why would it be beneficial to inhibit ENaC?

Answer 23

to try and compensate for loss of Cl- channel activity

Question 24

What other channels could they try and activate?

Answer 24

try and activate other cl- channels

Question 25

What % of activity do they need to be able to reach to restore lung function ?

Answer 25

> 20% CFTR ACTIVITY relative to normal is though to be enough to restore lung function

Question 26

What are CFTR potentiators?

Answer 26

drugs that stimulate CFTR activity- basically CFTR agonists

• stimulate ion channel activity but it only does this in certain circumstances- cant treat class 2 because these channels are not in the plasma membrane
• so only works for mutations that dont affect trafficking and synthesis of the protein

Question 27

What is an example of a CFTR potentiator?

Answer 27

IVACAFTOR (VX-770)
- drug now used clinically
works with type 3 (low basal activity), type 4 (smaller currrents) and type 5 (fewer channels in the membrane)
but this is only about 5% of CF patients
due to the low number of patients that this benefits, pharma companiesdont want to invest

Question 28

Where are many of the mutations ?

Answer 28

many mutations in the nucleotide binding domain so this may affect binding of ATP and hydrolysis of ATP

Question 29

What have electrophysiology recordings shown about CFTR mutations ?

Answer 29

CFTR mutations reduce channel activity- however IVACAFTOR is able to increase activity of mutant channels to level equivalent to WT behaviour

Question 30

What are CFTR correctors?

Answer 30

treatment of type 2 mutations which is most patients
also known as pharmacoloogical chaperones - somehow correct the shape, synthesis or trafficking of the molecules to the plasma membrane
- improves maturation and export of the mutant CFTR from the ER

Question 31

How are CFTR correctors thought to work ?

Answer 31

thought to work by allosteric stabilisation or by blocking degradation
- many appear to be allosteric modulators which bind to the protein and stabilise the shape to enable protein to pass through quality control and then reach the plasma membrane

Question 32

What did VX-809 do ?

Answer 32

increased CFTR maturation and chloride secretion in cultured F508 delta-HBE

• on SDS-PAGE F508 delta migrates further because it has a lower molecular weight therefore mature protein has a higher molecular weight
• VX809 increases the amount o f mature glycosylated protein which increases the amount of cl-channels present in the cells

Question 33

What doesn’t F508 do ?

Answer 33

doesn’t go to the golgi complex where a lot of the post translational modifications occur

Question 34

What does phosphorylation of CFTR do ?

Answer 34

it increases cl- channel activity

Question 35

What are type 1 mutations ?

Answer 35

nonsense, frameshift, splicing errors- minority of cases
5-10% of CF cases
they introduce premature stop codon and produce truncated protein

Question 36

What do amino glycoside antibiotics do ?

Answer 36

e.g. gentamicin
interfere with mRNA translation and cause errors in amino acid incoorporaton or peptide termination
- has an unusual side effect in ribosomes- can miss out stop codons an carry on reading the rest of the sequence

Question 37

What is PTC124 (Ataluren) used for ?

Answer 37

used in other disorders and can be used to treat any nonsense mutation disorder
-skips over the stop codon- as its concentration increases it causes the production of full length CFTR and improves lung function- less coughs per hour

Question 38

What treatment could be useful for type 3 mutants?

Answer 38

increasing cAMP levels
- one study did this by disrupting CFTR-NHERF2-LPA2 complex
- cftr is present in the plasma membrane with there scaffold proteins(nherf2) and LPA2(GPCR)
LPA2 is coupled to Gi and therefore activation during lung inflammation leads to less cAMP
want to disrupt the interaction of CFTR with NHEFR2 and LPA2 so there is less association - not use therapeutically yet

Question 39

What LPA2 drug was tested and what occurred?

Answer 39

CO-068 was incubated and it increased CFTR function by preventing LPA2 reducing adenyl cyclase activity
- they proved this worked by applying a CFTR inhibitor

Question 40

What other CL- channels could be activated to try and improve function ?

Answer 40

other epithelia cl- channels:

• clc-2 (lubiprostone) used in ibs
• calcium-activated chloride chanel (TMEM16A)- could be activated by stimulating intracellular calcium agents such as p2y agonists to increase calcium levels

Question 41

What do small molecule activators of tmem16a do ?

Answer 41

stimulate epithelial Chloride secretion and intestinal contraction

Question 42

What is Eact and T16Ainh and what did they do with them?

Answer 42

Eact= TMEM16A acitivator
T16Ainh= TMEM16A inhibitor
looked at fluid secretion in cultured bronchiolar epithelial cells
- secretion can be inhibited by T16Ainh therefore indicatingg that TMEM16A channels are important in this process