Small molecules treating CF - 1

Question 1

Current treatments for CF - what is the problem?

Answer 1

Reverse the symptoms but dont actually target the protein

Question 2

Current treatments for CF that alleviate symptoms?

Answer 2

Inhaled bronchodilators - opens up the airways
Nebulised antibiotics - fights infection
Mycolytics - enzymes break down mucus
Nebulised hypertonic saline - sets up driving force for movement from basolateral to apical side, diluting ,mucous and increasing height of the PCL
Physio and exercise - clears mucus
Steroids - fight inflammation
Fat soluble vitamins - allow patient to absorb vitamins properly
Pancreatic enzymes - break down food

Question 3

What is the main problem with treatment of CF in terms of different patients

Answer 3

Different mutations and classes of mutations for different patients.
dF508 most common (trafficking)
Regional mutations also apply with regards to mutation frequency

Question 4

What does VX-770 molecule target?

Answer 4

Potentiator molecule !
Targets the G551D mutation (gating) - channel doesn't open. 
Effects 1-3% CF patients 
Glycine > Aspartate 
severe symptoms!

Question 5

What does VX-809 molecule target?

Answer 5

dF508 mutation - protein not trafficked to membrane, 90% patients with CF have this
Phenylalanine deletion

Question 6

DRUGS SCREEN - G551D mutation cell based flourescence assay – what did it involve?

Answer 6

Load cells with fluorescent component – colour changes when the Vm of cells changes (due to the action of the CFTR channel)
Identified VX-770 as this caused the change of colour when added to cells

Question 7

DRUGS SCREEN - dF508 - cell based immunoblot assay – what did it involve?

Answer 7

CFTR is glycosylated to traffick to the membrane –> run along gel seeing how many mature and immature bands produced
Mutant cells –> have more immature band
Adding VX-890 shifted more cells from immature to mature band!

Question 8

Impacts of VX-770 on CF cells (rat thyroid)

Answer 8

Rat thyroid cells (decreases validity of results)
In mutant, when cells stim with cAMP (which stimulates CFTR), little increase in short circuit current seen
Adding VX-770 caused SSC to increase almost to WT level

Question 9

Impacts of VX-770 on CF cells (CF patient cells)

Answer 9

with cAMP stimulator and VX-770, 48% of WT response restored –> enough to alleviate symptoms!!!
Increased ciliary beat frequency
Increased height of liquid layer

Question 10

VX-770 - clinical trials - what was observed?

Answer 10

Randomised double blind trial
Lung function improvement over 2 weeks
Enhanced channel functioning when cAMP stimulator added
Sweat [Cl-] drops below the clinical threshold
Increased number of event-free patients

Question 11

VX-809/dF508 screen - cell immunoblot assay results

Answer 11

Ratio of immature:mature bands measured –> only 50% seen in mutant cells, but ratio increased upon addition of VX809 !!
Increased conc of drug = increased short circuit current corresponding to Cl- secretion

Question 12

PULSE CHASE EXPERIMENT - What does the procedure involve?

Answer 12

Expose HUMAN EMBRYONIC KIDNEY cells to radioactive componants, there are incorporated into cells and therefore CFTR being made (radioactivelly labelling CFTR)
Took away radioactive components and looked at cells for % mature and non-mature protein based on radioactive labelling

Question 13

PULSE CHASE EXPERIMENT - What did the results show?

Answer 13

Mutant cells + VX-809 had more mature band present (not quite same as WT)
Enough present to push protein to be trafficked to the membrane!!