Transcript Novel Potentiators Augment Efficacy of Translational

Novel Potentiators Augment Efficacy of Translational Readthrough in CFTR
Nonsense Mutations
Venkateshwar Mutyam1, Xiaojiao Xue1, David M. Bedwell1, Timor Baasov2, Martin Andrews3,
Steven van der Plas3, Katja Conrath3, Steven M. Rowe1
1CFRC, University of Alabama at Birmingham, Birmingham, AL., 2Technion-Israel Institute of Technology, Haifa, Israel, 3Galapagos NV, Generaal De Wittelaan L11A3, 2800 Mechelen, Belgium.
Combination approaches: GP-2 activity in FRT G542X cells
1.0
Δ FSK
2.0
A
0.8
Vehicle
VX-770
VX-809
GP-2
VX-809+GP-2
G418
G418+VX-809
G418+GP-2
G418+VX-770
G418+VX-809+VX-770
G418+VX-809+GP-2
1.5
1.0
HBE G542X: Effect of VX-770
8
0.6
0.4
0.2
0.5
0.0
0
10
20
30
40
50
G418
VX-809
VX-770
GP-2
60
-
+
-
+
+
-
A
Inh
VX-770
Forsk
B
4
FRT
WT
G542X
G542-C
3
0.4
0.2
0.0
1.0
0.5
0.0
1
10
100
1000
10000
Max efficacy
D
3
2
100
1000
10000
****
0.03
0
20
30
40
50
60
70
80
90
0
10
20
30
0.6
0.4
0.2
VX-770
Test compound (10 mM)
EC50
GP-2 VX-770
1476 2744
D
Δ FSK
Δ VX770
2
****
1
**
****
****
0
60
70
80
Δ FSK
Δ GP-2
****
2
FRT
WT
G542X
G542W
-
+
-
1.0
+
****
VX-770
Test compound (10 mM)
GP-2 VX-770
4248 26013
4
2
Vehicle
NB124
C
D
- Vehicle
+ NB124 treated
8
**
4
2
NB124
G418 treated HBE DF508/G542X
VX-770 vs. GP-2
NB-124 treated HBE DF508/G542X:
VX-770 vs. GP-2
+
Vehicle
G418 treated
****
6
4
*
2
-
+
-
0
+
GP-2 (10 mM)
-
+
VX-770 (10 µM)
-
+
GP-2 (0.1 µM)
-
****
****
G542R
G542C
****
G542R
0
G542C
FRT
WT
G542X
G542W
 The novel potentiator GP-2 is highly efficacious towards enhancing CFTR function following
translational RT of PTCs, and compares favorably with ivacaftor, even during chronic
administration.
FRT G542 amino acid variants
GP-2 vs. VX-770
Δ GP-2
0.8
 The combination approach of a RT drug with potentiators may boost nonsense suppression to
therapeutic levels of CFTR function.
0.6
 The combination of RT agents and potentiators deserves exploration in human subjects with
nonsense mutations.
0.4
0.2
FRT
WT
G542X
G542W
G542R
+
GP-2 (1 µM)
Conclusions
****
****
Acknowledgements
0.0
Fig.1. Effect of potentiators on FRT cells with stopcodon mutants pretreated with G418 : FRT cell monolayers
with PTC constructs (G542X, W1282X, R1162X) were pretreated with G418 for 48 hrs followed by conductance (Gt)
measurements with acute addition of FSK, potentiators, and CFTRInh-172 (A) Dose response (ranging from 3 nM to
10,000 nM) of GP-2, VX-770 in FRT G542X cells and (B) FRT W1282X cells. Scatter plots representing maximum
efficacy of potentiators at 10 μM in (C) FRT G542X cells and (D) FRT W1282X cells. (E) Enhanced CFTR activity in
the presence of 10 μM potentiators in FRT R1162X cells. In all the three PTC mutant cell lines, GP-2 is most efficacious
potentiator compared to VX-770. Potentiators had no effect on FRT parental cells without CFTR (data not shown)
indicating specificity. **** P<0.0001
**
****
1
Δ VX770
GP-2
Δ FSK
6
Fig. 4. Effect of GP-2 vs. VX-770 in primary human bronchial epithelial cells (ΔF508/G542X). HBE cells
were pretreated with RT agents (NB124 or G418) for 48 hrs followed by Isc measurements. (A) FSK+ VX770 &
(B) FSK+GP-2 stimulated Isc in NB124 pre-treated cells. (C) GP-2 induced Isc was significantly greater in
NB124 pre-treated cells compared to VX-770 (P<0.05) (D) G418 pre-treated HBE cells also exhibit significant
increase in CFTR potentiation even at 0.1 µM and 1 µM and GP-2 was been shown to be efficacious potentiator
compared to VX-770
* P<0.05, ** P<0.001, **** P<0.0001
90
0.01
0.5
EC50
3
****
0.02
VX-770
GP-2
Vehicle
*
0.00
1.0
50
Time (Mins)
1.5
0.0
GP-2
3
40
Δ GP-2
0
0
VX-770 (10 mM)
0
E
2.0
2
0
2
1
10
G542W
G542R
1
0
Inh
GP-2
****
2.5
Conductance (mS/cm2)
Conductance (mS/cm2)
10
Max efficacy
0.8
0.0
1
Concentration (nM)
Concentration (nM)
C
0.04
1.5
+
+
+
****
VX-770
2.0
+
+
+
-
FRT
WT
G542X
G542C
G542-R
Conductance (mS/cm2)
VX-770
0.6
GP-2
+
+
-
4
G542-W
0.05
D Conductance (mS/cm2)
GP-2
C
FRT R1162X cells: 10 mM Potentiators
+
+
Isc(mA/cm2)
Forsk
Conductance (mS/cm2)
2.5
Conductance (mS/cm2)
Conductance (mS/cm2)
0.8
FRT W1282X cells
+
+
-
8
4
GP-2 activity on FRT G542 amino acid variants
Conductance (mS/cm2)
FRT G542X cells
+
-
HBE G542X: Effect of GP-2
Δ VX770
Δ FSK
6
6
Time (Mins)
E
+
+
-
Fig. 2. Effect of chronic GP-2 treatment on FRT G542X cells. (A) Representative conductance (Gt) tracings of FRT
cell monolayers (n=3) expressing CFTR mutant G542X pretreated with G418 in combination with potentiators ( VX-770
or GP-2) or correctors (VX-809) for 48 hrs (B) FSK levels of different treatment combinations. G418 induced
readthtrough CFTR activity is significantly enhanced in the presence of VX-809 and VX-770 or GP-2 as compared to
VX-809 or VX-770 or GP-2 alone. In this combination therapy, GP-2 induced potentiation is comparatively higher than
VX-770.
Dose response of GP-2 & VX-770 in FRT cells with
CFTR PTC mutations following readthrough
B
+
+
Time (Mins)
Conductance (mS/cm2)
METHODS
0.0
Conductance (mS/cm2)
Cell Culture
• Fisher Rat Thyroid (FRT) cells: FRT’s were stably transduced with CFTR G542X, R1162X and W1282X stop
codons, along with matched CFTR WT control cDNA using the Flp-In system (Invitrogen).
To study the functional consequences of amino acids (AAs) inserted during PTC suppression, FRT CFTR
missense mutations with AA substitutions at the 542 position (Arg,G542A; Trp, G542W; Cys, G542C) were
created.
• Primary cells: Human bronchial epithelial cells derived from lung explants carrying a nonsense alleles G542X
in trans with F508del CFTR (G542X/ΔF508) were seeded on to permeable supports, grown in differentiating
media for at least 6-8 weeks until terminally differentiated.
Conductance (transepithelial chloride conductance, TECC) Assay
• For conductance (Gt) measurements cells were seeded on costar 24 well 0.4 uM permeable supports (~4.5
million cells/well) and maintained at 37°C with 5% CO2. Cells were grown to confluence and treated G418 or
NB124 (250 µg/ml) for 48 hrs.
• CFTR activity was measured as a change from baseline conductance following the addition of forskolin (20
µM), potentiators (GP-2, GP-4, VX-770; 10 µM) and then CFTRInh-172 (10 µM).
Ussing chamber
• Short circuit current (Isc) in primary cells was measured using this voltage clamped apparatus, where baseline
measurement was taken before the apical addition of 100 µM Amiloride. A chloride gradient was then imposed
by replacing the apical ringer’s solution with a low chloride Ringer’s with simultaneous addition of amiloride.
• FSK was administered to stimulate CFTR activity before Ivacaftor (VX-770) followed by the addition of CFTR
specific inhibitor, CFTRInh-172.
A
B
Isc(mA/cm2)
Methods
B
Isc(mA/cm2)
•We hypothesize that novel CFTR potentiators alone and in combination with translational RT could improve CFTR
activity and function to confer a significant therapeutic benefit to CF patients with PTCs.
A
Conductance (mS/cm2)
•CFTR potentiator ivacaftor (VX-770) was successfully used in restoring CFTR function in G551D and other related
mutations and has also been shown to augment CFTR function following RT by potentiating rescued CFTR
channels. However, ivacaftor may have deleterious effects on CFTR stability, thus the evaluation of alternative
potentiators is warranted.
Inh
FSK
Conductance (mS/cm2)
•Premature termination codons (PTCs) in the CFTR gene result in nonfunctional CFTR protein and are the
proximate cause of ~11% of CF causing alleles. Use of aminoglycosides has been shown to induce readthrough
(RT) of PTCs, restoring CFTR expression and function.
GP-2 activity in primary cells pre-treated with
readthrough agents
Isc(mA/cm2)
Introduction
G542C
Fig. 3. Effect of GP-2 vs. VX-770 on FRT G542 amino acid variants. Representative Gt tracings of FRT monolayers
expressing AA variants along with WT, subjected to acute treatment with (A)VX-770 or (B)GP-2. Summary of Gt
induced (C)FSK+ VX-770 and (D)FSK+GP-2. The FSK induced CFTR activity was further enhanced by addition of
potentiators (VX-770 or GP-2) and was significantly higher in WT, G542W, G542R and G542C compared to FRT (no
CFTR) cells. (E) GP-2 was more efficacious as a potentiator compared to VX-770
* P<0.05, **** P<0.0001
• UAB Cystic Fibrosis Research Center (CFRC)
• Galapagos NV