L4 - CF and Small Molecules I Flashcards
What is the CF gene product?
A Cl channel in the apical membrane
What is the structure of the CF Cl channel?
12 transmembrane spanning domains - 2 groups of 6 - Between each group is the regulatory domain Two nucleotide binding domains - NBD1 and NBD2 - Important in the gating of the channel
What is CFTR phosphorylated by?
PKA
In the regulatory domain
Plays an important role in the opening and closing of CFTR
Where are lots of CFTR mutations found?
NBD1 and NBD2
What is the most common CFTR mutation?
Delta F508
Where is the Delta F508 CFTR mutation found?
In NBD1
in 70-90% of individuals
What are the 6 CFTR mutation classes?
1 - Null production 2 - Trafficking 3 - Regulation 4 - Conduction 5 - Partial reduction mRN~a 6 - High turnover CFTR Mutations can belong to multiple classes depending on its impact on CFTR
Class I mutation - null production
mRNA from gene product is unstable
No CFTR made
Class II mutation - trafficking
CFTR protein is made but is not trafficked effectively to the membrane
E.g. F508
- Made but misfolded - meaning cell targets the protein for degradation rather than trafficking it to membrane
Class III mutation - regulation
CFTR is made and trafficked to membrane but is not regulated appropriately
Normally activation by PKA mediated phosphorylation - with mutation this has no effect
Or this phosphorylation does not occur
- Get differences in channel open probability
Class IV mutation - conduction
CFTR is made and trafficked to membrane but it does not open as it should do
The way the protein responds to regulation is not correct
Open probability of channel is lower than it should be
Class V mutation - partial reduction mRNA
mRNA is made but the amount is reduced
Reduced amount of CFTR
Class VI mutation - high turnover CFTR
Amount of time the CFTR spends at the apical membrane is reduced
High turnover of CFTR from cell membrane
E.g. F50D
- Doesn’t traffic to membrane properly and when it gets there it is removed quickly
What is the diagnostic threshold for sweat Cl concentration?
If a patient has severe CF their sweat Cl is typically above 60mmol/L Cl
In clinic one of the first tests is take a sweat sample and measure Cl
If your sweat chloride is 100 mmol what class mutation are you likely to have?
Classes 1-3
Very low CFTR function
Pancreases doesn’t work
If your sweat chloride is 80 mmol what class mutation are you likely to have?
Classes 4-5
10% CFTR function
Tend to have pancreatic sufficiency
- Don’t secrete digestive enzymes from pancreases because of CFTRs role in bicarbonate secretion
What is Congenital bilateral aplasia of the Vas Deferens ?
2 CF mutations
- Less severe mutations
Issue – inconclusive sweat test as sweat Cl below 60mmol
- Therefore may not get access to CF treatments
What is the next step if you get an inconclusive sweat test?
Genetic tests - but a lot of work
Nasal potential difference experiments
Biopsy of gut and looked at response to Ach
- Lost even in mild CF patients
What % normal CFTR protein do you need to get normal CFTR function?
50%
Some studies have even suggested 15%
How is Cl handled in the upper airway?
NKCC1 on basolateral membrane
- Brings Cl ions in using the Na gradient
CFTR opens in apical membrane
- Loss of Cl from the cell
- This drives water movement between the cells
- Sets height of ASL
How is Cl handling altered in the upper airway in CF patients?
Non-functioning CFTR Less Cl and water secretion Height of periciliary layer drops Cilia bend Thick mucus not cleared Airways problems/infection
How do ENaC and CFTR interact in the upper airway?
When CFTR is active, ENaC is inhibited and vice versa
Cell type specific
How does ENaC and CFTR interaction affect Na and Cl handling in the upper airways in a CF patient?
In CF CFTR in non-functional –> extra ENaC function
Reduced Cl secretion
Enhanced Na absorption
Exacerbates the impact on the ASL - height drops even further
How is Cl handled in the alveolar?
Movement of Cl reversed compared to upper airways
K/Cl cotransporter on basolateral membrane
- Removes Cl using K gradient
- Intracellular Cl is low
If you open CFTR
- Driving force is for Cl enter into cell
- Net absorption of Cl from ASL
How do ENaC and CFTR interact in the alveolar?
CFTR activates ENaC
Reabsorption of Cl and Na drive water reabsorption
- Height of ASL is optimum for gas exchange
What does CF cause in alveolar cells?
Often alveolar oedema
Impacts on their ability to get oxygen into their body
Why is salty sweat characteristic of CF patients?
Due to CFTRs role in absorbing Cl ions from lumen of sweat gland
CFTR independent secretion of Cl into lumen of sweat gland
Whatever is in the lumen is then lost at the skin
How is Na and Cl loss at the skin in CF patients vs normal individuals?
In normal individuals
- Distal sweat gland absorbs Cl and Na from the sweat
In CF
- CFTR non-functional don’t get absorption of Cl
- ENaC non-functional don’t get absorption of Na
How do ENaC and CFTR interact in the distal sweat gland?
CFTR activates ENaC
In distal sweat glands on what membrane is CFTR found?
Both apical and basolateral membrane
- High Cl on apical –> driving force for Cl to enter
- Low Cl on basolateral
- Lowest Cl in lumen –> driving force for Cl to leave
What are the current treatments for CF?
Nebulised antibiotics – tobramycin –> fight infection
Inhaled bronchodilators –> open airways
Mycolytics – pulmozyme –> breakdown mucous
Nebulised hypertonic saline (conc NaCl) –> hydrates ASL, improves mucocilary clearance
Oral antibiotics –> fight infection
- Issue in CF patients with antibiotic resistance
Pancreatic enzymes –> breakdown food
Fat soluble vitamins –> help absorb sufficient vitamins
Steroids –> decrease inflammation
Exercise and physiotherapy –> help clear mucous
High energy supplements –> helps absorb sufficient nutrients
What pharmaceutical company developed several clinically approved drugs to treat CF?
Vertex pharmaceuticals
What 4 drugs did Vertex pharmaceuticals develop?
Ivacaftor
Lumacaftor
Tezcaftor
Elexacaftor
What kind of drug are Lumacaftor, Tezcaftor and Elexacaftor?
Correctors
They traffic mutant CFTR to the membrane
What kind of drug is Ivacaftor?
A potentiator
Increases open probability of the channel
- Useful for mutations with gating defects
- E.g. G551D
How did they test the function of Ivacaftor?
Measures Isc in rat thyroid cells expressing either wild type CFTR or mutant (G551D) CFTR
What does Forskolin do?
Activates PKA –> phosphorylation of CFTR –> increased CFTR function
What happened when Forskolin was added to rat thyroid cells expressing wild type CFTR?
Large currents
What happened when Forskolin was added to rat thyroid cells expressing mutant CFTR?
Current double that in unstimulated
But only 10% of current seen in wild type with Forskolin
What happened when Forskolin and Ivacaftor was added to rat thyroid cells expressing mutant CFTR?
Large currents
But still only 40% of current seen in wild type with Forskolin
Shows Ivacaftor increases the open probability of mutant channels
What happened when Forskolin, Ivacaftor and CFTR inhibitor was added to rat thyroid cells expressing mutant CFTR?
Small current
Showed that the increase in current seen with Forskolin and Ivacaftor is mediated by CFTR
What did they do to test if Ivacaftor worked in cells from a patient?
Took HBE cells from patient expressing G551D and F508
Added amiloride to block ENaC
Added Forskolin
- Small increase in Isc
Added different concentrations of Ivacaftor
- As concentration goes up as does the Isc
- This is mediated by the mutant CFTR being stimulated by the Ivacaftor
Why did they add amiloride when they were looking at the CFTR protein not ENaC?
Changes in ENaC would have contaminated the recordings
After testing Isc to look at the function of Ivacaftor what other data did they look at to test VIP?
ASL volume and cilia beat frequency
What is VIP?
Aasoactive intestinal peptide
Used to stimulate PKA –> activateV CFTR
What happened to the height of the ASL in the presence of VIP or Ivacaftor?
Non-CF in presence of VIP –> height drops to optimum –> 40 uL
CF in presence of VIP –> height drops below optimum
CF in presence of VIP and Ivacaftor –> height in between WT and mutant
Ivacaftor shifts ASL height close to WT
What happened to the cilia beat frequency in the presence of VIP or Ivacaftor?
Mutant cells and VIP –> small increase
Mutant cells and Ivacaftor –> large recovery of open probability of CFTR
Mutant cells, VIP and Ivacaftor –> nearly full recovery of open probability of CFTR
Bringing the height of the layer back up helps return cilia beat frequency to normal
What did a randomised double blind and placebo controlled trial with Ivacaftor show?
Monitored FEV1 - expressed data as change in % of predicted FEV1
Baseline data showed both groups only had 64% lung function to begin with –> already suffering
Placebo – slow deterioration in lung function
Ivacaftor – within 2 weeks lung function improved by 10%
- This is maintained for the duration of the trial
What is pulmonary exacerbation?
Infection
More coughs
Poorer lung function measurement
Loss of weight
In the randomised double blind and placebo controlled trial with Ivacaftor what % of patients had pulmonary exacerbation?
Placebo - 41% of patients have no problems
Ivacaftor – 67% of patients have no problems
In the randomised double blind and placebo controlled trial with Ivacaftor what were the sweat chloride results?
Placebo – retain 100mmol Cl
Ivacaftor – drops below clinical threshold
What is Cl free isoprotenerol?
Cl free solution
Beta receptor agonist
Activates CFTR via cAMP
Generate a large Cl gradient –> large Cl secretion if CFTR active –> big shift in nasal transepithelial potential
What did the nasal transepithelial results show as you increases the concentration of Ivacaftor?
Cl channels now functioning - negative shift in potential
This is the experiment were they used Cl free isoprotenerol
An in vitro study examined the potential value of a new small molecule treatment in CF. Mutant CFTR mediates Isc was 17.5 uA in the absence, and 20.7 uA in the presence of the compound. From what we have discussed in the module so far is there value in taking this compound further?
CFTR mediated Isc means enhanced function
Increase is 20.7-17.5 = 3.2uA
This is an 18% increase
Studies suggest 15% increase in function is enough to alleviate the symptoms in CF patients yes worth taking further
Although this is in vitro – doesn’t always translate into clinical effectiveness
