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BMS346 > CF > Flashcards

CF Flashcards

1
Q

What is cystic fibrosis?

A 
most common lethal genetic disease in caucasions 
exocrine pancreatic insufficiency 
increased sweat Cl 
male infertility 
airway disease= biggest killer
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2
Q

Which leaky epithelial is capable of secreting Nacl rich fluid?

A 
exocrine gland acini
small intestine 
choroid plexus 
sweat gland coil
upper airway
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3
Q

What is an important model tissue for this

A

Shark rectal gland
secrete large amount of NaCl and is very robust
Can be left in baths for long time compared to rabbit/ rat

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4
Q

What are ouabain, barium and furosemide?

A 
Ouabain= Na/K ATPase blocker 
Barium= K channel blocker 
Furosemide= Blocks NKCC
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5
Q

Why is Oubain used to manipulate cl secretion present in extracellular compartment?

A

sets up df for Na influx across the basolateral membrane through NKCC DF goes down blocked

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6
Q

What does barium set up?

A

Reduce DF for Na influx

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7
Q

If Na/K ATPase what is the effect on Cl secretion?

A

Cl secretion drops to nothing
critical for Cl secretion
sets up driving force to maintain low intracellular sodium and neg potential

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8
Q

Barium blocker

A

depolarise potential, shifts from neg to 0

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9
Q

Furosemide

A

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

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10
Q

Cl is above electrochemical equilibrium

A

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

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11
Q

Cl= 17nM

A

that’s what we got but its actually 70mM- ICF

so there is an active process accumulating cl inside so its above electrochemical equilibrium

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12
Q

Why is it important to be above the electrochemical equilibrium

A

for it to get out all you need is open an cl on apical membrane and DF for ions to leave the cell

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13
Q

Cl channels on the apical membrane

A

Let ions leave
cAMP activated to open cl channel
stimulate conductance and overall secretion
furosemide blocks NKCC, lowers cl conc down to 17mM

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14
Q

What is Cl channels important for?

A

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

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15
Q

Cystic fibrosis gene product in Cl channel in the apical membrane

A

F508- 80% of patients with this mutation

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16
Q

Structure of CFTR

A

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

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17
Q

Nucleotides in CFTR

A

critical for function, lots of mutations in them

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18
Q

Mutations classes

A
  1. null production- mRNA unstable, breaks down, no protein made
  2. trafficking- not taken to membrane, protein sent for degradation
  3. regulation- PKA not normal, doesn’t open as much as it should
  4. conduction- gating mutation , channel doesn’t open and close
  5. partial reduction in mRNA- some mRNA not normal
  6. High turnover- channels made, trafficked but cell membrane is reduced
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19
Q

What does the type of mutation determine?

A

the severity

and the amount of sweat chloride

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20
Q

How does the amount of functioning CFTR affect sweat Cl?

A

CFTR protein function higher= lower amount of sweat chloride
*> 60 mmol diagnostic cut off for CF- above and you have CF

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21
Q

If you have 20-40mmol

A

Then it is inconclusive whether or not you have CF

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22
Q

What is congenital bilateral absence of the vas deferens

A

different classification- changed due to the severity

range of symptoms different even in some populations

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23
Q

Classes of vas deferens

A 
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
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24
Q

CFTR looked at in the colon

A

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

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25
Q

Diarrhoea

A

normal functioning CFTR that secrete lots of Cl

disrupting CFTR blockages down from GI tract

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26
Q

Cl secretion DF

A

Open up on apical membrane- Cl secretion

Open K channel on the apical membrane- froms and driving force for Cl secretion= hyperpolarises

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27
Q

Ach and pge2

A

ACH=Rise in IC Ca, activate apical and basolateral CL channel
increase secretion
PGE2= increase cAMP, stimulates secretion and CFTR

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28
Q

Effect of Carbachol CCH

A

is a Ach receptor- activation

increases Ca- IC

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29
Q

Effect of indomethacin

A

inhibits PG production

decreases cAMP

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30
Q

IBMX effect

A

inhibits phosphodiesterase

increases cAMP

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31
Q

Forskolin

A

activation of adenylate cyclase

increase cAMP

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32
Q

Looking at effects on Non-CF rectal tissue Cl secretion

method

A
  • enac blocked to stop contamination

- stimulate Cl secretion and get neg shift in VTE

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33
Q

If CFH is added to stimulated Cl secretion and in the presence of indomethacin

A

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

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34
Q

What happens if you add IBMX

A

Increase cAMP, bypassing receptor stimulating directly so donesnt matter if PGE is blocked by Indomethacin there is still increase in CL secretion

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35
Q

Effect of CGH, indomethacin and IBMX on Cl secretion

A

No effect in CF patients as CFTR is not functional so no Cl can be secreted

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36
Q

The CF colonic mucosa

A

Reduction/ abolishment of Cl secretion
meconium ileus ~ 10% newborns
M1 equivalent in adults- obstruction in small or large intestine

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37
Q

Problems arising drom CF

A

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

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38
Q

CFTR model

A

Cl leaving apical side
Enac apical membrane absorbing Na

basolateral
- 3Na out and 2K in
-NaKCC- in
Water moves out increasing fluid layer

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39
Q

Alveolar model

A

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

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40
Q

Distal sweat glands model

A

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

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41
Q

Why does CF have salty sweat

A

due to problem with NaCl reabsorption

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42
Q

What is the future treatment for CF

A

Small molecules

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43
Q

Symptoms of CF

A 
depletion of PCL 
failure to clear mucous 
infection 
inflammation 
tissue damage 
decrease lung function- not enough normal tissue to support life
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44
Q

Function of setting up a DF

A

for Na absorption of Cl secretion

height go down and cilia to bent to beat

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45
Q

Current treatments

A

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

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46
Q

new drug developments for cystic fibrosis

A

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
47
Q

Two drugs so far

A

VTX-770- ivacaftor/ kalydeco*
VTX-809- corrector
*licensed for G551D carrying proteins

48
Q

Regional variation in mutation frequency exists

A

F508 90% allelic frequency worldwide

  • 50% Spain
  • 80% northern Spain
49
Q

Drug screen for G551D

A 
Glyane to aspartate
Type 3 
1-3% 
severe symptoms 
gating defects
screening- based on fluorescence vm assay 228,000 chemical tested 
- VX-770
50
Q

F508 drug screen

A 
phenylalanine deletion 
type II 
90%
severe symptoms 
trafficking defect 
screening- cell based immunoblot assay mature CFTR levels 164,000 chemicals tested 
-VX-809
51
Q

F805 screening test

A

whether it was going to membrane- when it does it becomes glycosylated and you see 2 bands
lots of immature lower band

52
Q

VX770 and G551D

A

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)

53
Q

Looked at G551D Mutant in the presence of PKA and ATP and stimulate phosphorylation

A

downward deflection means cftr channels are opening
Add FSK= small increase
add VX770 get channels open most of the time - big deflection

54
Q

Looking at expression system in native tissue not rats- G551D

A

human bronchial airway cells
complex mutation- got 2 (G551D + another)
add amiloride and over time add FSH and increase dose

55
Q

HBE cells G551D and F508 response

A

wt fsh response- 56
G551D + F508- 5% of wt
G551D+ F508 + VX-770 - 48% of wt

56
Q

Use these response want to measure height of asl in vitro

A

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

57
Q

Cilia beat frequency

A

measure how fast they beat

- cilia bent as layer down so cant beat fast

58
Q

affect of 770 on CBF

A
  • VX770 increase CBF

- 770 + VIP (combine with cAMP)= restored to normal level

59
Q

VX770 and G551D clinical trial

A
  • randomised, double blind, placebo
    83 CF patients with atleast 1 mutation- 150mg/day for 48 weeks
    = monitored FEV1 as a % of predicted
60
Q

Patients in the 770 drug trial

A

Already have compromised lung

started at the similar height and weight

61
Q

2 weeks using the ivacftor

A

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

62
Q

Looking at the effect of using different concentrations of 770

A 
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
63
Q

glycosylation of patients with F508 mutation

A

Immature glycosylated- remains immature and it is sent for degradation due to misfolding

64
Q

Experiments using VX809 and F508

A

fisher rate expressing f508 CFTR

interested in compounds that shift from immature to mature

65
Q

Ratio of mature to total

A

No vertex 808= 0.5 mature

increase VX809 conc- more mutant protein and 30% now mature

66
Q

Exposing cells to VX809

A

it inserts into protein
correct misfolding
get trafficked to the membrane as normal

67
Q

Plus chase experiment HEK and CFTR

A
  1. methianina/ cysteine
  2. new mature CFTR produced 35S labelled
  3. 35S compounds removed and cells left for different durations
  4. some CFTR glycosylated- mature and some 35 CFTR non-glycosylated = immature
  5. ratio of these depend on how long cells left and pharmaceutical manipulation cells exposed to VX809
68
Q

Western blot for CFTR, F508+vehicle, F508 +vx809

A

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

69
Q

HBE patients -/- for F508

A 
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
70
Q

Dose reponse in -/- F508 patients

A

response bigger when dose is bigger

71
Q

Clinical trial - F508 and VX809

A 
- randomised, double blind, placebo 
18-19 CR patients 
homozygous for F508 
different amounts 
28 days
72
Q

Results from the VX809 clinical trial

A

Used FEV1
no pattern of activity
lines different concs- highest conc has fall in sweat
chloride only 6mmol- still above clinical threshold

73
Q

Combination drug

A

770 + 809= combotherapy

74
Q

Why would they license combination drug

A
  1. change in sweat cl= neg value= -15 mmol
    - 250mg has largest fall, bigger than VX alone but does not go below threshold
  2. FEV1 change- placebo goes down as lung function falls, steadily worsening. 600 mg vx809 and ivacaftor 250- dramatic improvement
  3. full clinical trial phase 3
    - 1108 patients
    - homozygous for F508
    - over 12 years old
75
Q

Results of full clinical trial phase 3 combination drugs

A

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

76
Q

The data from this combined trial suggest 3 things

A
  1. VX809 can traffic F508 cftr to membrane and is functional
  2. F508 patients VX809 alone not sufficient to improve symtpoms
  3. VX809 traffics 508 CFTR to membrane and 770 potentiate enhance
77
Q

Downside of the combination therapy

A

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

78
Q

Gene therapy

A

1990s treatment to change CF was gene therapy and it hasn’t

79
Q

Reasons for gene therapy not working

A

Cells replaced - asthma inhaler- but airway cells are replaced and new cells come that don’t have WT CFTR

80
Q

First clinical trial for non viral gene therapy 2015

A

62 patients- placebo or PG169/GL67A

81
Q

Results of gene therapy trial

A
  1. airway function stabilised
    - hasn’t worsened/improved
    - patient with good lung function can stabalise high
  2. forrest plot
    - FEV1= positive impact
    - FVC= lung volume improved
    - gas trapping signicantly reduced
  3. change in bronchial DNA score
    - most able to take up coding sequence of CFTR
    - increase Cl secretion
    - largest shift in pot seen
82
Q

What does gene therapy data suggest

A
  • stabilises lung function
  • +/- population- respond well
  • could be due to liposome formulation
  • step forward but more work needed
83
Q

What happens to patients with 2 faulty copies of CFTR?

A

drop in function of CFTR and increase sweat cl

84
Q

Classical CF

A

sever symptoms
majority 2 mutations CFTR
1-2% northern Europe 2
mutations NOT observed

85
Q

Atypical CF

A

Mild symptoms
1 or No CFTR symptoms
carriers typically no symptoms

86
Q

Hypothesis for Enac mutated airway

A

GOF mutation
depletion ASL
CF like symptoms

87
Q

Enac genes screened- 31 patients

A 
a- SCN1A- 11 mutations 
B- SCN1B- 7 mutations 
Y-SCN1Y- 8 mutations 
* additional mutations observed 
took mutants expressed in cells and viewed
88
Q

Impact of mutants on enac function

A

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

89
Q

Work in patients to validate GOF epithelial ion channel

A
  1. CF
  2. Atypical- should be carrier of CFTR, Enac mutation
  3. normal subject vte- 125mv, squirt in low cl solution shift= 16mv
90
Q

Add amiloride to see function of Enac

A

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

91
Q

Summary of Enac

A
  1. some GOF mutations in Enac in patients
  2. these could explain cf like symptoms
  3. some GOF mutations in Enac
  4. how do these impacts lead to CF like symptoms?
92
Q

aw493R mutation

A

Found in EC loop of subunit

increase enac current= GOF

93
Q

Study to confirm AW493R function

A

Add amiloride- bigger function of Enac when wash out amiloride sensitive current is increased with mutant

94
Q

Na feedback inhibition

A

Enac opens- na floods in - enac endocytosed into apical membrane- enac retrived from the membrane

95
Q

Feedback Na in mutants

A

shouldn’t see much affect on currents as continue to see high sodium currents as disrupt endocytosis

96
Q

Na feedback inhibition and AW493R

A

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

97
Q

Cleaved channels

A

Cleaved Enac (proteases)- higher Po

98
Q

Comparing cleaved and uncleaved enac

A

Uncleaved

  • silent
  • low Po
  • generate smaller enac

Cleaved

  • very high
  • high po
  • generate greater enac currents
99
Q

Response to chymotrypsin

A

Wt- add to ec surface
Enac cleaved
Po goes up
* know its enac bc when amiloride added it decreases

100
Q

Chymotrypsin effect on mutant

A

response to chymotrypsin lost
(thought it was bc channels are already cleaved)
- turns out gating of the channel was different and increased Po

101
Q

Pattern of activity of channel opening- effect from chymotrypsin

A

WT- flickering’s show channel open and close
- add chymotrypsin and it opens
Mutant- sustained open events, adding chymotrypsin has no effect

102
Q

Why does mutant have sustained opening events

A

reduced response to chymotrypsin not due to increase endogenous cleavage before its added

103
Q

Na self inhibiton

A

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

104
Q

Summary of Na Po

A
  1. AW493R increase Enac currents
  2. no change in Na feedback
  3. high currents Not increase cleavage
  4. loss of self Na inhibition
  5. high currents- increase water absorption- CF like
105
Q

BV348M mutation

A

B subunit of Enac
change in Po
wt- increase current when take away amiloride
mutant GOF-40-45% more current

106
Q

MTSET used

A

Sulfhydryl agent, binds cysteine, stabalises open state of channel
stabilises Po at ~1= measurer current before and after MTSET gives estimation of Po

107
Q

Examples of MTSET

A
  • amiloride sensitive current before MTSET- 12microamps

- amiloride sensitive current after MTSET- 20microamps

108
Q

What was used as background

A

Cysteine mutant

  • WT= BS52OC- Po= 0.24
  • BV348M- B3520C- Po=~0.33
109
Q

If you add cysteine that protein would work

A

No impact on Ca channel

GOF= Po bigger, more Na, water follows so height of liquid decreases

110
Q

Summary of Enac BV348M

A
  1. increase Enac currents
  2. No change channel number
  3. increase number Po
  4. high current, greater water absorption- Cf like symptoms
111
Q

Mouse overexpression of SCN1B (B subunit)

A

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

112
Q

Slice through lung of SCN1B mutant

A

Overexpressing plugs and plaques- lots of gas trapped as increase mucous

113
Q

Mice given intra-tracheal injection

A

Clearance of bacteria monitored after 3 days
wt- gone
Mutant- lots left- GOF Enac problem with clearing mucous

114
Q

Summary of SCN1B mutant

A
  1. B subunit overexpression increase Na current
  2. depletion ASL
  3. increase mucous plugs
  4. increase inflammation
  5. poor bacteria clearance
  6. CF like symptoms

BMS346 (5 decks)

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