L4 - CF and Small Molecules I Flashcards

1
Q

What is the CF gene product?

A

A Cl channel in the apical membrane

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2
Q

What is the structure of the CF Cl channel?

A
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
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3
Q

What is CFTR phosphorylated by?

A

PKA
In the regulatory domain
Plays an important role in the opening and closing of CFTR

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4
Q

Where are lots of CFTR mutations found?

A

NBD1 and NBD2

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5
Q

What is the most common CFTR mutation?

A

Delta F508

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6
Q

Where is the Delta F508 CFTR mutation found?

A

In NBD1

in 70-90% of individuals

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7
Q

What are the 6 CFTR mutation classes?

A
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
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8
Q

Class I mutation - null production

A

mRNA from gene product is unstable

No CFTR made

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9
Q

Class II mutation - trafficking

A

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

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10
Q

Class III mutation - regulation

A

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

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11
Q

Class IV mutation - conduction

A

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

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12
Q

Class V mutation - partial reduction mRNA

A

mRNA is made but the amount is reduced

Reduced amount of CFTR

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13
Q

Class VI mutation - high turnover CFTR

A

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

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14
Q

What is the diagnostic threshold for sweat Cl concentration?

A

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

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15
Q

If your sweat chloride is 100 mmol what class mutation are you likely to have?

A

Classes 1-3
Very low CFTR function
Pancreases doesn’t work

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16
Q

If your sweat chloride is 80 mmol what class mutation are you likely to have?

A

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

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17
Q

What is Congenital bilateral aplasia of the Vas Deferens ?

A

2 CF mutations
- Less severe mutations
Issue – inconclusive sweat test as sweat Cl below 60mmol
- Therefore may not get access to CF treatments

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18
Q

What is the next step if you get an inconclusive sweat test?

A

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

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19
Q

What % normal CFTR protein do you need to get normal CFTR function?

A

50%

Some studies have even suggested 15%

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20
Q

How is Cl handled in the upper airway?

A

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

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21
Q

How is Cl handling altered in the upper airway in CF patients?

A
Non-functioning CFTR 
Less Cl and water secretion 
Height of periciliary layer drops
Cilia bend  
Thick mucus not cleared
Airways problems/infection
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22
Q

How do ENaC and CFTR interact in the upper airway?

A

When CFTR is active, ENaC is inhibited and vice versa

Cell type specific

23
Q

How does ENaC and CFTR interaction affect Na and Cl handling in the upper airways in a CF patient?

A

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

24
Q

How is Cl handled in the alveolar?

A

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

25
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
26
What does CF cause in alveolar cells?
Often alveolar oedema | Impacts on their ability to get oxygen into their body
27
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
28
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
29
How do ENaC and CFTR interact in the distal sweat gland?
CFTR activates ENaC
30
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
31
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
32
What pharmaceutical company developed several clinically approved drugs to treat CF?
Vertex pharmaceuticals
33
What 4 drugs did Vertex pharmaceuticals develop?
Ivacaftor Lumacaftor Tezcaftor Elexacaftor
34
What kind of drug are Lumacaftor, Tezcaftor and Elexacaftor?
Correctors | They traffic mutant CFTR to the membrane
35
What kind of drug is Ivacaftor?
A potentiator Increases open probability of the channel - Useful for mutations with gating defects - E.g. G551D
36
How did they test the function of Ivacaftor?
Measures Isc in rat thyroid cells expressing either wild type CFTR or mutant (G551D) CFTR
37
What does Forskolin do?
Activates PKA --> phosphorylation of CFTR --> increased CFTR function
38
What happened when Forskolin was added to rat thyroid cells expressing wild type CFTR?
Large currents
39
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
40
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
41
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
42
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
43
Why did they add amiloride when they were looking at the CFTR protein not ENaC?
Changes in ENaC would have contaminated the recordings
44
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
45
What is VIP?
Aasoactive intestinal peptide | Used to stimulate PKA --> activateV CFTR
46
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
47
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
48
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
49
What is pulmonary exacerbation?
Infection More coughs Poorer lung function measurement Loss of weight
50
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
51
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
52
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
53
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
54
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