CAT-5571 AS A NOVEL AND POTENT AUTOPHAGY ACTIVATOR THAT
ENHANCES THE TRAFFICKING OF THE F508DEL-CFTR
Liu, F.1; Carney, D.1; Chandran, S.1; Lee, D.1; Lonkar, P.1; Nichols, A.1; Picarella, D.1; Ting, A.1; Webb, S.1; Wensley, A.1; Yeager, M.1; Bridges, R. J.2, PenaRasgado, C.2; Lacerda, A.3; Bordwell,
C.3; Vu, C. B.1 1. Catabasis Pharmaceuticals, Cambridge, MA, USA. 2. Chicago Medical School, North Chicago, IL, USA. 3. Charles River, Cleveland, OH, USA.
Introduction
Results
N
H
C A T -5 6 3 5 (3 µ M )
CAT-5635
CAT-5571
0 .2
DHA
SMART
Linker
C A T -5 6 3 5
V X -8 0 9
+ V X -8 0 9
+ V X -7 7 0
Defective CFTR
Enzyme-specific
cleavage of
SMART linker
ROS
Multi-node
pathway
modulation
Autophagy
F508del CFTR
aggresomes
C A T -5 6 3 5
C A T -5 6 3 5 /n o e n z y m e
0
Degradation of the
CFTR by the
proteasome
0
50
100
150
T im e (M in )
+ V X -8 0 9
-A T P a s e
*** #
0 .2 0
0 .5
0 .1 5
*
0 .1 0
0 .0 5
0 .0 0
2
V e h ic le
C A T -5 6 3 5
+ V X -7 7 0
+ V X -8 0 9
1
V X -8 0 9
+ V X -7 7 0
+ V X -7 7 0
Primary homozygous F508del hBE cells (Charles River Labs, patient code
KKCFFT006F) treated for 24 hrs with the above treatment groups.
Quantification of the immunoblots for the CFTR Band B, the CFTR Band C,
and the ratio of Band C to Band B. Error bars represent standard deviation;
SEM ± SEM, n = 3, * p < 0.05, ***p < 0.005, compared to Vehicle/VX770; # p
< 0.05, compared to the VX-809/VX-770 treatment group, with ANOVA
followed by Dunnett’s multiple comparison test.
CAT-5635 pre-incubated with VX-809 + VX-770
for 24 hrs at 37 oC
forskolin
27
VX-809 (3 µM) + VX-770 (0.1 µM)
Vehicle + VX-770 (0.1 µM)
H u m a n p la s m a
CAT-5635 (0.075 μM)
+ VX-809 (3 μM) + VX-770 (0.1 μM)
bumetanide
bumetanide
bumetanide
Vehicle + VX-770
VX-809 + VX-770
22
forskolin
forskolin
forskolin
R a t P la s m a
B e a g le P la s m a
CAT-5635 (0.15 μM) + VX-809 + VX-770
17
CFTRinh-172
12
2
100
50
40
B u m e ta n id e
)
V X -8 0 9
+ V X -7 7 0
2
)
*
(µ A /c m
8
6
10
4
2
C A T -5 6 3 5
+ V X -8 0 9
+ V X -7 7 0
Robert J. Bridges, homozygous F508del hBE, CFFT0018I
(SEM, n = 3). * p < 0.05, compared to VX-809/VX-770 with
Dunnett’s multiple comparison test.
*
600
400
200
0
V e h ic le
V X -8 0 9
V e h ic le
C A T -5 6 3 5
V X -8 0 9
+ V X -7 7 0 + V X -7 7 0
+ V X -8 0 9
C A T -5 6 3 5
V e h ic le
V X -8 0 9
C A T -5 6 3 5
+ V X -7 7 0
+ V X -7 7 0
+ V X -8 0 9
+ V X -8 0 9
+ V X -7 7 0
+ V X -7 7 0
Charles River, homozygous F508del hBE, KKCFFT006F
(SEM, n = 4). * p < 0.05, compared to VX-809/VX-770 with
Dunnett’s multiple comparison test.
A triple combination consisting of CAT-5571 enhanced the correction of the F508delCFTR relative to the VX-809/VX-770 combination
CAT-5571 pre-incubated with VX-809 for 24 hrs at 37 oC
CAT-5571 pre-incubated with VX-809 for 24 hrs at 37 oC
forskolin + VX-770
Vehicle
Vehicle
VX-809
Cmpd 5 (0.0375 μM)+ VX-809
VX-770
forskolin
VX-770
VX-809
CAT-5571 (0.018 μM) + VX-809
forskolin
VX-770
forskolin
Conclusion
bumetanide
bumetanide
bumetanide
CFTRinh-172
B u m e ta n id e
A U C
6
C F T R
800
in h
AUC
-1 7 2
2
)
(µ A /c m
2
*
400
200
3
2
Q
0
4
600
∆ IE
2
µ A /(4 5 m in * c m
)
*
4
2
(µ A /c m
∆ B u m e ta n id e
)
CAT-5571 represents a potential novel therapeutic approach for the treatment of
cystic fibrosis with the F508del mutation
800
0
0
+ V X -7 7 0
• CAT-5571 delivers a sustained exposure of CAT-5635, a covalent conjugate of
cysteamine and DHA
• CAT-5635 potently activates autophagy in cultured primary homozygous F508del hBE
cells at ≤ 3 µM. Neither cysteamine nor DHA (alone or in combination) activates
autophagy at the higher concentration (250 µM).
• CAT-5635 enhances the correction of the F508del-CFTR relative to the VX-809/VX-770
combination
• Expression of CFTR Band B/C
• CFTR chloride channel conductance
130
-1 7 2
)
+ V X -7 7 0
+ V X -8 0 9
20
+ V X -7 7 0 + V X -7 7 0
TECC-24: Primary homozygous F508del hBE cells were incubated in differentiation media at
37 oC for 24 hr. The media was then switched to Coon’s F12, with test compounds added
back. Plate was incubated at 37 oC for 4 hr prior to IEQ measurements. Representative IEQ
traces following the addition of benzamil (3 μM) to first block currents deriving from ENaC.
Forskolin (10 μM) was added and the IEQ was recorded over a 27 min period at 37 oC.
Bumetanide (20 μM) or CFTRinh-172 (20 μM) was added to block the chloride secretion
from the CFTR. Quantification of the bumetanide or CFTRinh-172 -inhibited CFTR chloride
current for the indicated treatment groups, ΔIEQ (μA/cm2). Quantification of the AUC
(computed for the time period that spans the IEQ value at the time of forskolin addition up
to the IEQ value at the time of bumetanide or CFTRinh-172 addition, min * μA/cm2).
120
µ A /(6 0 m in * c m
V e h ic le
+ V X -7 7 0
Methods
in h
Q
50
C A T -5 6 3 5
+ V X -7 7 0
Time (min)
2
)
100
0
+ V X -7 7 0
*
2
150
0
V X -8 0 9
110
100
90
10
∆ IE
1
30
*
200
(µ A /c m
2
V e h ic le
80
A U C
Q
3
T im e (h r )
70
C F T R
P e a k fo r s k o lin
250
∆ IE
)
2
*
µ A /(2 7 m in * c m
.0
.5
.0
.0
2
1
0
0
.0
.5
.0
.0
2
1
0
0
.0
.5
.0
2
1
0
.0
0
.0
.5
.0
2
1
0
0
.0
0
60
A U C
4
2
C A T -5 5 7 1 (% )
7
50
1
0
-2
V e h ic le
V X -8 0 9
(3 µ M )
C A T -5 5 7 1
(0 .0 3 7 5 µ M )
V X -8 0 9
V X -8 0 9
+ V X -7 7 0
1 .0
CAT-5635 pre-incubated with VX-809 + VX-770
for 24 hrs at 37 oC
M o u s e P la s m a
150
C A T -5 6 3 5
V e h ic le
+ V X -7 7 0
A triple combination consisting of CAT-5635 enhanced the correction of the F508delCFTR relative to the VX-809/VX-770 combination
CAT-5571 plasma stability
Autophagy activation by
the simultaneous
inhibition of TG2 and
activation of AMPK.
This reduces p62 and
allows the trafficking of
the CFTR to the cell
surface
1
Disease
pathway
p62
0 .1
2
(µ A /c m
p62
*
R a tio o f B a n d C /B a n d B
3
∆ B u m e ta n id e
p62
D H A (µ M )
Release of
bioactives
Cross-linking
Beclin 1
Hydrolysis of CAT-5635 using recombinant
Fatty acid amide hydrolase (FAAH)
3
AMPK
TG2
CAT-5635
N
H
0 .2
+ V X -7 7 0
*
Primary homozygous F508del hBE cells (Charles River Labs,
patient code KKCFFT004I) treated for 24 hrs at 37 oC with the
above treatment groups. Quantification of the immunoblots
shown. Error bars represent standard error mean (SEM, n = 3).
*p < 0.05, compared to the vehicle with ANOVA followed by
Dunnett’s multiple comparison test.
O
***
+ V X -7 7 0
IEQ (μA/cm2)
Cysteamine
-A T P a s e
0 .0
V e h ic le
0
HS
+
+
0 .0
0 .0
p 6 2 /a c tin (A .U .)
SMART linker conjugate
/ K
0 .4
p 6 2
A covalent conjugate of cysteamine
and docosahexaenoic acid (DHA)
/K
N a
0 .5
+
+
N a
1 .0
*
0 .6
0 .0
Working hypothesis
B a n d C /N a
-A T P a s e
B an d C /
A T P ase
+
1 .5
+
*
2 .0
1 .5
N
H
+
/K
B and B /
2 .5
L C 3 -II/L C 3 -I
HS
/K
0 .3
+ V X -7 7 0
O
+
0 .8
B e c lin -1
cysteamine-DHA metabolite
O
Enzymatic
degradation
B a n d B /N a
B a n d -B
O
O
c y s te a m in e (2 5 0 µ M ) + D H A ( 2 5 0 µ M )
B a n d -C /
H Me Me
N
S
S
c y s te a m in e (2 5 0 µ M )
D H A (2 5 0 µ M )
V e h ic le
V X -8 0 9
(3 µ M )
C A T -5 5 7 1
(0 .0 3 7 5 µ M )
V X -8 0 9
Robert J. Bridges, homozygous F508del hBE, CFFT0028H
(SEM, n = 3). * p < 0.05, compared to VX-809/VX-770 with
Dunnett’s multiple comparison test.
µA/(60 min*cm2)
N
H Me Me
N
SH
CAT-5635 increased CFTR Band-B and Band-C
in the presence of VX-809 and VX-770
v e h ic le
L C 3 -II/L C 3 -I (A .U .)
N
CAT-5635 activates autophagy in primary
homozygous F508del hBE monolayers
B e c lin -1 /A c tin (A .U .)
Autophagy is a physiological process that helps maintain cellular homeostasis through the
orderly degradation and recycling of dysfunctional cellular components. Depressed
autophagy has previously been reported in cystic fibrosis patients with the common F508del
mutation. Herein we describe the biological characterization of an autophagy activator, a
covalent conjugate between cysteamine and the omega-3 fatty acid docosahexaenoic acid
(CAT-5635), in primary homozygous F508del human bronchial epithelial (hBE) cells.
400
*
300
200
100
0
0
V e h ic le
V X -8 0 9
(3 µ M )
C A T -5 5 7 1
(0 .0 1 8 µ M )
V X -8 0 9
Vehicle
VX-809
(3 µM)
CAT-5571
(0.018 µM)
VX-809
Charles River, homozygous F508del hBE, KKCFFT0012I
(SEM, n = 4). * p < 0.05, compared to VX-809/VX-770 with
Dunnett’s multiple comparison test.
References
Acknowledgements
Mizushima et al. Autophagy: Renovation of Cells
and Tissues. Cell 2011, 147, 728-741.
We thank Dr. Martin Mense from the Cystic Fibrosis
Foundation Therapeutics for helpful discussion and
guidance. We also thank Dr. Steven Freedman
(Harvard Medical School ) for additional input.
Luciani, et al. Defective CFTR Induces Aggresome
Formation and Lung Inflammation in Cystic
Fibrosis through ROS-Mediated Autophagy
Inhibition. Nat. Cell Biol. 2009, 12, 863-877.