CF Flashcards
What is cystic fibrosis?
most common lethal genetic disease in caucasions exocrine pancreatic insufficiency increased sweat Cl male infertility airway disease= biggest killer
Which leaky epithelial is capable of secreting Nacl rich fluid?
exocrine gland acini small intestine choroid plexus sweat gland coil upper airway
What is an important model tissue for this
Shark rectal gland
secrete large amount of NaCl and is very robust
Can be left in baths for long time compared to rabbit/ rat
What are ouabain, barium and furosemide?
Ouabain= Na/K ATPase blocker Barium= K channel blocker Furosemide= Blocks NKCC
Why is Oubain used to manipulate cl secretion present in extracellular compartment?
sets up df for Na influx across the basolateral membrane through NKCC DF goes down blocked
What does barium set up?
Reduce DF for Na influx
If Na/K ATPase what is the effect on Cl secretion?
Cl secretion drops to nothing
critical for Cl secretion
sets up driving force to maintain low intracellular sodium and neg potential
Barium blocker
depolarise potential, shifts from neg to 0
Furosemide
Blocks NKCC co transport, Cl went to 0 as it directly inhibits influx- Cl get into cell through NKCC1 but other channels are essential
*All critical for normal Cl secretion
Cl is above electrochemical equilibrium
No active component coming in for Cl so the Cl in should be the same as extracellular
there is no active component
IC no active component
Cl= 17nM
that’s what we got but its actually 70mM- ICF
so there is an active process accumulating cl inside so its above electrochemical equilibrium
Why is it important to be above the electrochemical equilibrium
for it to get out all you need is open an cl on apical membrane and DF for ions to leave the cell
Cl channels on the apical membrane
Let ions leave
cAMP activated to open cl channel
stimulate conductance and overall secretion
furosemide blocks NKCC, lowers cl conc down to 17mM
What is Cl channels important for?
protein important for whole cl secretion process
apical membrane potential move to nerst cl when it needs to secrete Cl
so have net movement across epithelium
Cystic fibrosis gene product in Cl channel in the apical membrane
F508- 80% of patients with this mutation
Structure of CFTR
12TM domains
1 subunit makes channel and lots of other proteins that interact
divide into domain 1 and 2, get NBD1 1 and 2 binding sites for nucleotides to regulate how the channels open and close
Nucleotides in CFTR
critical for function, lots of mutations in them
Mutations classes
- null production- mRNA unstable, breaks down, no protein made
- trafficking- not taken to membrane, protein sent for degradation
- regulation- PKA not normal, doesn’t open as much as it should
- conduction- gating mutation , channel doesn’t open and close
- partial reduction in mRNA- some mRNA not normal
- High turnover- channels made, trafficked but cell membrane is reduced
What does the type of mutation determine?
the severity
and the amount of sweat chloride
How does the amount of functioning CFTR affect sweat Cl?
CFTR protein function higher= lower amount of sweat chloride
*> 60 mmol diagnostic cut off for CF- above and you have CF
If you have 20-40mmol
Then it is inconclusive whether or not you have CF
What is congenital bilateral absence of the vas deferens
different classification- changed due to the severity
range of symptoms different even in some populations
Classes of vas deferens
1-3= all CF with pancreatic insufficiency- sweat cl= 100mmol 4-5= 70-80, pancreatic sufficiency but have problems with organs and airways 6= not generated for the 2013 review
CFTR looked at in the colon
Isolate rat colonic crypt
lower to mid crypt cells cl secretion
CFTR on membrane of crypt - secrete ions and water follows - determine the water content of what you secrete
Diarrhoea
normal functioning CFTR that secrete lots of Cl
disrupting CFTR blockages down from GI tract
Cl secretion DF
Open up on apical membrane- Cl secretion
Open K channel on the apical membrane- froms and driving force for Cl secretion= hyperpolarises
Ach and pge2
ACH=Rise in IC Ca, activate apical and basolateral CL channel
increase secretion
PGE2= increase cAMP, stimulates secretion and CFTR
Effect of Carbachol CCH
is a Ach receptor- activation
increases Ca- IC
Effect of indomethacin
inhibits PG production
decreases cAMP
IBMX effect
inhibits phosphodiesterase
increases cAMP
Forskolin
activation of adenylate cyclase
increase cAMP
Looking at effects on Non-CF rectal tissue Cl secretion
method
- enac blocked to stop contamination
- stimulate Cl secretion and get neg shift in VTE
If CFH is added to stimulated Cl secretion and in the presence of indomethacin
increase intracellular Ca increasing Cl secretion
Indomethacin- blocks PGE reducing cAMP so reduce CFTR activity so decreases CL secretion even if you add CGH you don’t get secretion as you have DF but channels are closed
What happens if you add IBMX
Increase cAMP, bypassing receptor stimulating directly so donesnt matter if PGE is blocked by Indomethacin there is still increase in CL secretion
Effect of CGH, indomethacin and IBMX on Cl secretion
No effect in CF patients as CFTR is not functional so no Cl can be secreted
The CF colonic mucosa
Reduction/ abolishment of Cl secretion
meconium ileus ~ 10% newborns
M1 equivalent in adults- obstruction in small or large intestine
Problems arising drom CF
thick mucous- airway problems- respiratory tract infections and lung damage kills 70%
Enac interacts with CFTR- Enac function is enhance- increased Na and H20 absorption
CFTR model
Cl leaving apical side
Enac apical membrane absorbing Na
basolateral
- 3Na out and 2K in
-NaKCC- in
Water moves out increasing fluid layer
Alveolar model
increased fluid in alveoli
CFTR and Enac not working - no Cl and Na
open CFTR on apical and get Cl absorption
Basolateral side- no NKCC1, KCl co transport instead - moves Cl and K out of cell- Cl conc inside sits below equilibrium
water moves to basolateral in
Distal sweat glands model
Secrete from cells and then moves down usually absorbed again
Enac and CFTR on apical- absorption
basolateral- other epithelial Cl channels= ClC voltage gated channel, CaCC, Max1, 1cl, VOL
Water moves in
Why does CF have salty sweat
due to problem with NaCl reabsorption
What is the future treatment for CF
Small molecules
Symptoms of CF
depletion of PCL failure to clear mucous infection inflammation tissue damage decrease lung function- not enough normal tissue to support life
Function of setting up a DF
for Na absorption of Cl secretion
height go down and cilia to bent to beat
Current treatments
Nebulised antibiotics tobramycin- fight infection
inhaled broncholdilators- open airway
mycolytic pulmozyme- breakdown mucous
oral antibiotics- fight infection
pancreatic enzymes- breakdown food
fat soluable vitamins- absorb sufficient vitamin levels
steroids- decrease inflammation
exercise and physiotherapy- clear mucous
high energy supplements- help with sufficient nutrient absorption
nebulised hypertonic saline- hydrate ASL, improve muscocillary clearance
new drug developments for cystic fibrosis
vertex pharmaceuticals bought aurora bioscvences in 2001
- established drug screening programme
- aurora already had CF Programme
- vertex persuaded to continue it by VS-CF foundation
Two drugs so far
VTX-770- ivacaftor/ kalydeco*
VTX-809- corrector
*licensed for G551D carrying proteins
Regional variation in mutation frequency exists
F508 90% allelic frequency worldwide
- 50% Spain
- 80% northern Spain
Drug screen for G551D
Glyane to aspartate Type 3 1-3% severe symptoms gating defects screening- based on fluorescence vm assay 228,000 chemical tested - VX-770
F508 drug screen
phenylalanine deletion type II 90% severe symptoms trafficking defect screening- cell based immunoblot assay mature CFTR levels 164,000 chemicals tested -VX-809
F805 screening test
whether it was going to membrane- when it does it becomes glycosylated and you see 2 bands
lots of immature lower band
VX770 and G551D
fisher rat thyroid cells expressing wt and G551D CFTR
Fsk- stimulate cAMP
I’m at mouse this increases cl secretion but in the G551D mouse there is no increase
Added 770 to G551D and cl secretion increased by 12-15% ( proven it was CFTR as increase abolished when CFTR is inhibited)
Looked at G551D Mutant in the presence of PKA and ATP and stimulate phosphorylation
downward deflection means cftr channels are opening
Add FSK= small increase
add VX770 get channels open most of the time - big deflection
Looking at expression system in native tissue not rats- G551D
human bronchial airway cells
complex mutation- got 2 (G551D + another)
add amiloride and over time add FSH and increase dose
HBE cells G551D and F508 response
wt fsh response- 56
G551D + F508- 5% of wt
G551D+ F508 + VX-770 - 48% of wt
Use these response want to measure height of asl in vitro
Excess volume and height of layer falls over time
mutant CFTR exposed to 770 get some activity of CFTR channel as it opens up - secrete CL
*1/2 of wt and mutant
Cilia beat frequency
measure how fast they beat
- cilia bent as layer down so cant beat fast
affect of 770 on CBF
- VX770 increase CBF
- 770 + VIP (combine with cAMP)= restored to normal level
VX770 and G551D clinical trial
- randomised, double blind, placebo
83 CF patients with atleast 1 mutation- 150mg/day for 48 weeks
= monitored FEV1 as a % of predicted
Patients in the 770 drug trial
Already have compromised lung
started at the similar height and weight
2 weeks using the ivacftor
clear difference
improved and stayed improved
- PE, 67% haven’t in ivacafter and 41% haven’t in placebo
- ivacaftor patients have dropped below sweat threshold- no longer CF
Looking at the effect of using different concentrations of 770
Shift in Cl free isoprotenerol in nasl passage activate CFTR via cAMP Cl secretion= neg shift= higher dose= neg shift 25mg the worst and 150 the best
glycosylation of patients with F508 mutation
Immature glycosylated- remains immature and it is sent for degradation due to misfolding
Experiments using VX809 and F508
fisher rate expressing f508 CFTR
interested in compounds that shift from immature to mature
Ratio of mature to total
No vertex 808= 0.5 mature
increase VX809 conc- more mutant protein and 30% now mature
Exposing cells to VX809
it inserts into protein
correct misfolding
get trafficked to the membrane as normal
Plus chase experiment HEK and CFTR
- methianina/ cysteine
- new mature CFTR produced 35S labelled
- 35S compounds removed and cells left for different durations
- some CFTR glycosylated- mature and some 35 CFTR non-glycosylated = immature
- ratio of these depend on how long cells left and pharmaceutical manipulation cells exposed to VX809
Western blot for CFTR, F508+vehicle, F508 +vx809
WT- by 180mins everything is shifted to mature, goes to membrane. amount of radioactivity decreases in immature- still some being made but not radioactive
F508- immature disappears but little mature is made
VX809- immature disappears, slightly more mature appears
HBE patients -/- for F508
human bronchiole epithelial cells amiloride for Enac inhibition Cells exposed to cAMP stimulates PKC- small response add VX809 Response bigger as you increase the dose
Dose reponse in -/- F508 patients
response bigger when dose is bigger
Clinical trial - F508 and VX809
- randomised, double blind, placebo 18-19 CR patients homozygous for F508 different amounts 28 days
Results from the VX809 clinical trial
Used FEV1
no pattern of activity
lines different concs- highest conc has fall in sweat
chloride only 6mmol- still above clinical threshold
Combination drug
770 + 809= combotherapy
Why would they license combination drug
- change in sweat cl= neg value= -15 mmol
- 250mg has largest fall, bigger than VX alone but does not go below threshold - FEV1 change- placebo goes down as lung function falls, steadily worsening. 600 mg vx809 and ivacaftor 250- dramatic improvement
- full clinical trial phase 3
- 1108 patients
- homozygous for F508
- over 12 years old
Results of full clinical trial phase 3 combination drugs
1- 2 weeks FEV1 increase 3% well sustained in 600 and 400mg
- 24 weeks- subset of groups that work really well, moderate to no improvement (highest group only saw moderate improvement
2- forest plot- lie above black line then combo is better, below placebo is better- no difference in FEV1 shown
3- Development of cough or infection- placebo 40% and combo 20% had problem
4- hospitalisation- lots of placebo emitted and less combo
The data from this combined trial suggest 3 things
- VX809 can traffic F508 cftr to membrane and is functional
- F508 patients VX809 alone not sufficient to improve symtpoms
- VX809 traffics 508 CFTR to membrane and 770 potentiate enhance
Downside of the combination therapy
Cost
ivacaftor= £189,000 per patient per year- improvement in patients= don’t need other treatment*licensed
Orkambi= £104,000= Small impact, still need other treatments- benefit minimal
Gene therapy
1990s treatment to change CF was gene therapy and it hasn’t
Reasons for gene therapy not working
Cells replaced - asthma inhaler- but airway cells are replaced and new cells come that don’t have WT CFTR
First clinical trial for non viral gene therapy 2015
62 patients- placebo or PG169/GL67A
Results of gene therapy trial
- airway function stabilised
- hasn’t worsened/improved
- patient with good lung function can stabalise high - forrest plot
- FEV1= positive impact
- FVC= lung volume improved
- gas trapping signicantly reduced - change in bronchial DNA score
- most able to take up coding sequence of CFTR
- increase Cl secretion
- largest shift in pot seen
What does gene therapy data suggest
- stabilises lung function
- +/- population- respond well
- could be due to liposome formulation
- step forward but more work needed
What happens to patients with 2 faulty copies of CFTR?
drop in function of CFTR and increase sweat cl
Classical CF
sever symptoms
majority 2 mutations CFTR
1-2% northern Europe 2
mutations NOT observed
Atypical CF
Mild symptoms
1 or No CFTR symptoms
carriers typically no symptoms
Hypothesis for Enac mutated airway
GOF mutation
depletion ASL
CF like symptoms
Enac genes screened- 31 patients
a- SCN1A- 11 mutations B- SCN1B- 7 mutations Y-SCN1Y- 8 mutations * additional mutations observed took mutants expressed in cells and viewed
Impact of mutants on enac function
3,4,6,7- all different backgrounds
8,9,10- didn’t show anything might be mutation in different genes
1,2,5- decrease in Na
Work in patients to validate GOF epithelial ion channel
- CF
- Atypical- should be carrier of CFTR, Enac mutation
- normal subject vte- 125mv, squirt in low cl solution shift= 16mv
Add amiloride to see function of Enac
Atypical carriers -25mv, low cl solution shows shift to -15
mutant Enac similar to CFTR mutant
enac mutation and mild CFTR not far from normal but increased Enac shift with amiloride
Summary of Enac
- some GOF mutations in Enac in patients
- these could explain cf like symptoms
- some GOF mutations in Enac
- how do these impacts lead to CF like symptoms?
aw493R mutation
Found in EC loop of subunit
increase enac current= GOF
Study to confirm AW493R function
Add amiloride- bigger function of Enac when wash out amiloride sensitive current is increased with mutant
Na feedback inhibition
Enac opens- na floods in - enac endocytosed into apical membrane- enac retrived from the membrane
Feedback Na in mutants
shouldn’t see much affect on currents as continue to see high sodium currents as disrupt endocytosis
Na feedback inhibition and AW493R
At high and low Na- number of channels in membrane @ start= no difference
rise in current as more Na available which activates endocytosis
WT low Na== wait for a bit then enac pulled out of membrane
High Na= ratio of % drop in wt same as mutant
Mutant- Na feedback inhibition the same, got more enhanced- not anything to do with lack of endocytosis
Cleaved channels
Cleaved Enac (proteases)- higher Po
Comparing cleaved and uncleaved enac
Uncleaved
- silent
- low Po
- generate smaller enac
Cleaved
- very high
- high po
- generate greater enac currents
Response to chymotrypsin
Wt- add to ec surface
Enac cleaved
Po goes up
* know its enac bc when amiloride added it decreases
Chymotrypsin effect on mutant
response to chymotrypsin lost
(thought it was bc channels are already cleaved)
- turns out gating of the channel was different and increased Po
Pattern of activity of channel opening- effect from chymotrypsin
WT- flickering’s show channel open and close
- add chymotrypsin and it opens
Mutant- sustained open events, adding chymotrypsin has no effect
Why does mutant have sustained opening events
reduced response to chymotrypsin not due to increase endogenous cleavage before its added
Na self inhibiton
When Na goes in
decreases in channel Po
Add 100mM Na-influx into cell- inhibition from sodium self
mutant- no fall, self inhibition lost even when Na added
Summary of Na Po
- AW493R increase Enac currents
- no change in Na feedback
- high currents Not increase cleavage
- loss of self Na inhibition
- high currents- increase water absorption- CF like
BV348M mutation
B subunit of Enac
change in Po
wt- increase current when take away amiloride
mutant GOF-40-45% more current
MTSET used
Sulfhydryl agent, binds cysteine, stabalises open state of channel
stabilises Po at ~1= measurer current before and after MTSET gives estimation of Po
Examples of MTSET
- amiloride sensitive current before MTSET- 12microamps
- amiloride sensitive current after MTSET- 20microamps
What was used as background
Cysteine mutant
- WT= BS52OC- Po= 0.24
- BV348M- B3520C- Po=~0.33
If you add cysteine that protein would work
No impact on Ca channel
GOF= Po bigger, more Na, water follows so height of liquid decreases
Summary of Enac BV348M
- increase Enac currents
- No change channel number
- increase number Po
- high current, greater water absorption- Cf like symptoms
Mouse overexpression of SCN1B (B subunit)
Atypical CF- overexpress B subunit
rate limiting step in enac function
add this makes lots of channels- PCL layer reduced in mutant in both bronchus and trachea
Slice through lung of SCN1B mutant
Overexpressing plugs and plaques- lots of gas trapped as increase mucous
Mice given intra-tracheal injection
Clearance of bacteria monitored after 3 days
wt- gone
Mutant- lots left- GOF Enac problem with clearing mucous
Summary of SCN1B mutant
- B subunit overexpression increase Na current
- depletion ASL
- increase mucous plugs
- increase inflammation
- poor bacteria clearance
- CF like symptoms
