New insights in

peripheral quality control of CFTR

Tsukasa Okiyoneda
Department of Biomedical Chemistry, School of Science and
Technology, Kwansei Gakuin University, Japan
The CFTR mutants are eliminated from the PM by peripheral QC
Reduced the efficacy of CF drug
CF pathogenesis (class 6)
Cl- Cl- Peripheral
PM Quality Control

unstable !
PM destabilized
CFTR class 6 mutants
N287Y (MSD1)
R347P (MSD1) CFTR stabilizer lysosome
S492F (NBD1)
-Improve the current drug efficacy
r∆F508 (NBD1)
A561E (NBD1)
L1077P (MSD2)
-develop the drug for CF patients
carrying class 6 CFTR mutations degradation
W1282X (NBD2)
N1303K (NBD2)
Q1411X (CT)
4326∆ITC (CT)
4279insA (CT) Fukuda & Okiyoneda, Front Pharmacol. 2018
4271∆C (CT)
 Molecular mechanism of the peripheral CFTR QC
Cl- Cl-
PM Peripheral Quality Control
r∆F508 Ub Ub
CFTR Ub Ub
Ub
Ub
Hsc70
Ubiquitination
CHIP

1. Ubiquitination facilitates the CFTR elimination from the PM.

Sharma M et al, J Cell Biol 2004, Glozman R & Okiyoneda T et al, J Cell Biol 2009

2. CHIP (chaperone-associated Ub ligase) determines

the ubiquitination for the CFTR peripheral QC.
Okiyoneda T et al, Science 2010, Fu L et al, PLoS One 2015
lysosome

CHIP (STUB1) is a drug target for the CFTR stabilizer ? degradation

 CHIP Knockout has a deleterious effect

CHIP KO mice decrease longevity with

accelerated aging phenotypes.

CHIP KO mice exhibits

an accumulation of misfolded proteins and
a decrease in proteasome activity.

Counteracting CHIP activity is not

a preferable therapeutic approach for CF.

Min JN et al,
Mol Cell Biol. 2008 Jun;28(12):4018-25.
CHIP KD partially inhibits the rΔF508 elimination from the PM
Mature r∆F508 CFTR stability Only partially inhibit
r∆F508 the CFTR elimination.
CHX (h): 0 2 4 6
100

% remaining C form
siNT C
B 75
C
siHsc70 B 50

25
C
siCHIP
B
0
Okiyoneda et al, Science 329, 805, 2010

r∆F508 CFTR
PM

Ub Ub
Hsc70 ? Peripheral
Quality Control
Ub Ub
Ub
lysosome
Ub
CHIP
Other Ub ligases could be involved ! degradation
 A comprehensive Ub ligase siRNA phenotypic screening
responsible for the PMQC of CFTR
r∆F508 CFTR PM density in CFBE cells
HRP activity
CFTR specific = CFTR PM level
E3 candidates
relative rΔF508 CFTR level (vs siNT)

HRP HRP
∆F508 CFTR
-HRP (peroxidase)
RFFL
CF bronchial epithelia (CFBE)

26°C rescue &

37°C, 2.5 h chase

siRNA library
(636 Ub ligases)

96 well plate
relative CD4T PM level (vs siNT)
(a stable PM model protein) Okiyoneda T et al, Dev Cell 2018
 RFFL (CARP2: RNF189: RIFIFYLIN)
 FYVE-like and RING finger domain containing E3 Ub ligase
RFFL FYVE-like RING 363 aa

RFFL cellular localization (HeLa)

RFFL-GFP RFFL-GFP
mRFP-Rab5 (EE) mRFP-Rab7 (LE)

RFFL-GFP
RFFL-GFP RFFL-GFP
mRFP-Lamp1 (L) WGA (PM)

RFFL-ΔNT-GFP R. Sakai (D3)

PM & endosome localization Okiyoneda T et al, Dev Cell 2018

 RFFL selectively reduces the PM level of unfolded CFTR
PM density of PM cargo in CFBE cells (ELISA)

siNT siRFFL
600 CD4
PM

Ub λm
500 αHA C (mature) Lamp1
(rΔF508) tetra- misfolded
PM density (% of siNT)

B
mono-Ub
400
αRFFL

αNa/K
300 ATPase

lysosome
200
recycling lysosome lysosome recycling
100

0
ccUb
rΔF508 WT T70 Lamp1 λm Transferrin
(AllRΔG) Receptor
unfolded unfolded

CFTR CD4T
(model proteins)

Okiyoneda T et al, Dev Cell 2018

 RFFL facilitates rΔF508 CFTR endocytosis & lysosomal degradation
Endocytosis in 5 min (ELISA) Metabolic stability of rΔF508 CFTR (CFBE)

CFBE siNT Rescued ΔF508 (26°C)

40
siRFFL siNT siRFFL
endocytosis (%)

CHX (h) 0 1 2 3 0 1 2 3
30
αHA C (mature)
(rΔF) B
20

10 post-Golgi ER
Mature (band C) Immature (band B)

0 100
siNT
100
siNT

Remaining CFTR (%)

Remaining CFTR (%)

rΔF508 TfR CD4TccUb siRFFL siRFFL
unfolded (AllRΔG) 75 75

PM 50 50
rΔF508 TfR
Tetra- 25 25
RFFL mono-Ub
AP-2 0 0
KD
(clathrin) Epsin1 0 1 2 3 0 1 2 3
Eps15 CHX chase (h) CHX chase (h)

endocytosis
Okiyoneda T et al, Dev Cell 2018
 RFFL effect is not limited to rΔF508 CFTR & airway cells
Class VI mutant (CFBE-tet) Cell type
293MSR (embryonic kidney)
siNT siRFFL 200 ∗

mature rΔF508 (%)

αHA C 150
(rΔF508) B
100
αATPase
50
αRFFL RFFL

0
siNT siRFFL

CFPAC-1-tet (pancreas)
200

rΔF508-3HA PM density
∗
150

(% of siNT)
100

50
RFFL KD increased the PM stability of T70 & P67L CFTR
(class VI mutants). 0
siNT siRFF
The RFFL effect is not specific to airway (CFBE) cells L
because siRFFL increased cell surface rΔF508-CFTR
in 293MSR & CFPAC-1 cells
Okiyoneda T et al, Dev Cell 2018
 RFFL interacts with rΔF508 CFTR at the PM & endosomes
Selective interaction with unfolded CFTR BiFC
HBH-CFTR: - r∆F WT (Biomolecular Fluorescence Complementation)

RFFL-V5: - + - + - + interaction
GFP-V5: + - + - + -

RFFL

RFFL
rΔF
ΔF
48 RFFL
Avidin -V5 VN VC Venus
pull-down
35
WB: αV5 Venus mRFP-Rab5 (EE)
(RFFL) 25

WB: αHA 180

(HBH-CFTR) C (mature)

Venus
RFFL
-V5 Interaction at
endosome & PM
Input 35
WB: αV5
(RFFL)
25
(kDa) GFP
M. Miyata -V5 R. Sakai (D3)

RFFL selectively interacts with misfolded rΔF508-CFTR

Okiyoneda T et al, Dev Cell 2018
 RFFL interacts with mature rΔF508 CFTR by the disordered regions
RFFL-HB (His-Biotin) RFFL-HB (His-Biotin)
Pull-down

C5610A
experiment

∆NYAP
∆FYVE

∆RING

∆2-10

1-120
1-148
1-200
1-220
∆DR1

∆DR2

∆DR3

mock
mock

∆CID

1-38
1-88
∆CT

WT
WT
(kDa) (kDa)
PD: NA-agarose C
WB: αHA (r∆F) C 140
140 (mature)
(mature)
55
55 40
35
WB: HRP-NA 40
(RFFL) 20
35
15
BHK cells 10
RFFL 1 148
mutants WT BHK cells
∆DR1
∆FYVE unfolded
∆DR2 PM, rΔF508 CFTR
∆NYAP endosome
∆DR3
∆CID
∆CT disordered
∆RING (DR1, DR2)
18-39 97-106 162-232 298-313
Disordered
Regions 119-147 248-256 363
(PONDR®)
DR1 DR2

Okiyoneda T et al, Dev Cell 2018 R. Sakai (D3)

 RFFL facilitates r∆F508 CFTR ubiquitination in the post-Golgi
CFTR Ub level rΔF508 Ub chain
(ELISA) Post-Golgi (mature r∆F508)

ΔF K48-Ub level K63-Ub level

CFTR-Ub level (% of siNT)

- 120 120
100 100
Avidin (kDa) Ub-
pull-down CFTR 80 P<0.01 80
250
60 60
WB: αUb 180
(P4D1) 40 40
P<0.01
135 20 20
Relative Ub level: 1.0 0.25 0 0
siNT siRFFL siNT siRFFL
C (mature)
αHA 180
(CFTR) B ER (immature ∆F508)
rΔF508 Ub level (stable KD) K48-Ub level K63-Ub level
CFTR-Ub level (% of siNT) 120 120
100 N.S.
CFTR-Ub level (%)

100 N.S.
100
80
80 80
60
60 60
40 P<0.01
40 40
20
20 20
0
siNT shRFFL 0 0
siNT siRFFL siNT siRFFL
Okiyoneda T et al, Dev Cell 2018
RFFL directly ubiquitinates unfolded CFTR (cytoplasmic NBD1)
CFTR ubiquitination in vitro CFTR NBD1 ubiquitination in vitro
- BHK rΔF WT NBD1 (1S): ΔF508 WT
Lysate:
E1/UbcH5c/RFFL:
E1/UbcH5c/RFFL:
denature (°C): 30 30 37 44 30 30 37 44

210
(kDa) Ub- 140
NA pull-down (CFTR)

CFTR 90 Ub-
αUb 210 NBD1
70
(P4D1) αNBD1 55
140 40
NBD1
35
(kDa)
unfolded unfolded
αHA
210
(CFTR) C (mature) RFFL: WT Δ1-148 H333A
140
E1/UbcH5c:
denature (°C): 37 37 44 37 44 37 44
210
αUb 140
sup

(P4D1) 90 210
140
70
90 Ub-
55
αNBD1 70 NBD1
(ΔF1S) 55
folded unfolded
40
Ub NBD1
Δ1-148 35
(ΔDR1&2) (kDa)
Okiyoneda T et al, Dev Cell 2018
 RFFL KD improves the limited efficacy of CF drug
ΔF508 expression (CFBE) PMQC
Cl- rΔF508 CFTR
VX-809 (unfolded) system
siRNA: NT RFFL PM

C (mature) Not effective.. CFTR

αHA elimination
(ΔF) B
VX-809
(CF drug)
αATPase

Lysosome
ΔF508 function (YFP quenching assay)
ER
1000 siNT P<0.01 degradation
ΔF508 function (% of NT)

siRFFL
RFFL KO mice
- No abnormal phenotype
(Ahmed AU et al, Curr Biol 2009)
500

RFFL could be a CF drug target !

RFFL inhibitor : CFTR stabilizer
0
DMSO VX-809 (first-in-class CF drug)
PROPHYLACTIC OR THERAPEUTIC AGENT FOR
RFFL KD improves the CF drug efficacy (>2-folds). CYSTIC FIBROSIS (Okiyoneda, PCT/JP2018/009524)

Okiyoneda T et al, Dev Cell 2018 Fukuda & Okiyoneda, Front Pharmacol. 2018
 AlphaLISA (GST-RFFL & His-sumo-NBD1 direct interaction)
RFFL & ΔF508 CFTR-NBD1 PPI (protein protein interaction)

(kDa)
GST- His-
RFFL ΔF508 NBD1
70 ー
55 ー
40 ー
Donor beads
Acceptor beads
1 hr
mix
2 hr (RT) 1O
2
Ex Em
680 nm 615 nm

Donor Acceptor
Bead Bead

The AlphaLISA is very simple, and easily measures the direct interaction in vitro.
 AlphaLISA chemical library screen (Bioactive 1758 compounds)

GST-RFFL (7.5 nM) & myc-ΔF508-NBD1 (20 nM)

1758 compounds
Z’ factor >0.5, S/B > 4.8
>50% inhibition GST-RFFL
100 Myc-NBD1
90
80 83 compounds
Percentage inhibition

70
Reproducibility GST-RFFL
60 Myc-NBD1
50 TruHit assay
40
12 compounds
30
20 Reproducibility GST-RFFL
10 in KG Univ His-NBD1

0
0 500 1000 1500 Compound A
Compound number

Collaboration with ASUBIO PHARMA CO., LTD. unpublished data

 Compound A is the RFFL-ΔF508 NBD1 PPI inhibitor
RFFL-ΔF508 NBD1 PPI (AlphaLISA)
7000
50 nM RFFL & 75 nM ΔF508-NBD1
6000

Compound A & its analog A’ inhibit the PPI

Alpha signal （a.u.）

5000
in a concentration-dependent manner.
4000

3000

2000

1000

0
- DMSO 1 3 10 50 2 10 (μM)

Compound A A’
(analog)
Y. Ono (B4) unpublished data
 Compound A & A’ act as the rΔF508 CFTR stabilizers
rΔF508 CFTR rΔF508 CFTR
PM stability (CFBE cells) PM level (CFBE cells) rΔF508-CFTR
∗∗
Remaining PM CFTR after 1 h（%）

200 PM
∗∗
60 ∗∗
∗∗ compound
A
50 150

% of DMSO
40 ubiquitination

100
30

20
50
10

0 0
Lysosomal degradation
DMSO A A’ DMSO A A’
(5 μM) (2 μM) (5 μM) (2 μM)

Compound A & A’ increases PM stability & PM level of rΔF508 CFTR.

unpublished data
 Compound A improves the CF drug efficacy on ΔF508 CFTR function
Apical ΔF508 CFTR current (CFBE cells) CFTR Inhibitor-172
Sensitive current

CFTR 16
24
+VX-809 (CF drug)

ΔF508 CFTR current （μA/cm2）

Inh-172
14
22
12
20
Current （μA/cm2）

10
18 Genistein
8
16
6
14 Forskolin
A (5 μM) 4
12
2
10
DMSO
0
8 DMSO A
900 1100 1300 1500 1700 1900
(5 μM)
Time (sec)

Compound A increased the ΔF508 CFTR current induced by VX-809,

and achieved almost two-fold improvement of the CF drug efficacy.

R. Fukuda unpublished data

 The RFFL inhibitor could be a CFTR stabilizer
Peripheral
r∆F508 CFTR
PM Quality Control

Ub Ub
Ub Ub lysosome
Ub
compound A Ub
Hsc70 (PPI inhibitor) K63-linked poly-Ub

CHIP degradation
siRFFL

・RFFL directly and selectively recognizes unfolded CFTR (e.g., rΔF508 CFTR)
& facilitates the ubiquitination for the peripheral QC.

・RFFL inhibitors (siRFFL, PPI inhibitors) improve the PM stability of unfolded CFTR
& the CF drug efficacy (e.g., Orkambi) .

The RFFL inhibitor could be a CFTR stabilizer as a first-in-class of

CF drug which improves the efficacy of Orkambi and may provide
the clinical benefit for CF.
 Remaining issues
 RFFL inhibition can improve rΔF508 CFTR functional expression
in CF-HBE (primary culture model) and CF animal model ?

 DUB (deubiquitinase) & other Ub ligases are involved in the

CFTR peripheral QC ?

Peripheral
r∆F508 CFTR Quality Control
PM

Ub Ub
Hsc70 Ub Ub lysosome
Ub
E3 ? Ub
CHIP DUB ?

Targets for CFTR stabilizer ? degradation

We need to uncover the whole of the peripheral QC mechanism to develop

the CFTR stabilizer for the robust CF pharmacological therapy
 Acknowledgements
Okiyoneda Lab member
Ono Sakai Cystic Fibrosis Translational
(B4) (D3)
Research Centre
Department of Physiology
Gergely L. Lukacs
Guido Veit

EMBL Australia
Fukuda Pirjo Apaja

ASUBIO PHARMA CO., LTD.

We are looking for pharmaceutical companies as collaboration partners

to develop the CFTR stabilizer (e.g., RFFL inhibitor).

Please contact : t-okiyoneda@kwansei.ac.jp

 RFFL may limit ΔF508-CFTR PM expression under physiological conditions

rΔF508 CFTR-HRP PM level CFTR mRNA level (q-PCR)

The RFFL KD effect was still observed as an increased rΔF508-CFTR-HRP PM level in

CFBE-teton cells, where the exogenous CFTR-HRP mRNA level was adjusted to a lower
level of the endogenous CFTR mRNA in Calu-3 cells.

Okiyoneda T et al, Dev Cell 2018

 Highly correlated ΔF508-CFTR functional correction
between CFBE cell lines & CF-HBE

Veit G et al, Nature Medicine 2018

RFFL mRNA expression in CF bronchial epithelial cells
q-PCR (CFBE-tet) q-PCR (primary culture)

RFFL gene expression is not upregulated

by ΔF508-CFTR expression or in primary
human bronchial epithelial (HBE) cells from
CF patients carrying the ΔF508 mutation.
 2018 Oct 8

Triple combination (3C)

・VX-809 (type I)
・#4172 (type III, NBD1 bind)
・#3151 (type II, C4-like)
 ΔF508 CFTR PM stability upon the 3C treatment
Mature ΔF508 CFTR stability （CFBE cells）

3C

3C

rΔF508 CFTR is still eliminated upon the 3C treatment !

Veit G et al, Nature Medicine 2018

 RFFL KD enhances the 3C effect in CFBE cells
ΔF508 CFTR-HRP PM level

2000 Cl- rΔF508 CFTR

siNT **
1800
siRFFL PM
1600
CFTR
1400
% of siNT-DMSO

elimination
1200
3C
1000 (CF drug)
800

600 Lysosome
ER
400 **
** ** degradation
200

0
DMSO VX-809 Orkambi 3C

RFFL inhibitor could be an option

to improve the 3C efficacy.
DMSO: 0.3% for 1 day
VX-809: 3 μM
Orkambi: 3 μM VX-809 & 100 nM VX-770
3C: 3 μM VX-809, 3 μM 3151, 3 μM 4172
unpublished data
CFBE-tet-ΔF508-HRP cells
 CFTR mutations and CF drug

T70

Corrector (Lum)
Stabilizer
Drug & Potentiator (Iva)
Not available
Potentiator (Iva)

Lopes-Pacheco M. Front Pharmacol. 2016 Sep 5;7:275 (modified)

RFFL KD inhibits lysosomal sorting of rΔF508 CFTR from endosomes
Endocytosed rΔF508 CFTR (CFBE) 37°C 1 h label (αHA)
siNT rΔF508 EEA1 DAPI siRFFL rΔF508 EEA1 DAPI

chase chase
0h 0h

chase chase
2h 2h

Lysosome accumulation of rΔF508 CFTR (CFBE) M. Aki (M2) 0,5

shNT
0,4 shRFFL

rΔF rΔF 0,3 P<0.01

PCC
0,2

shNT Lysotracker shRFFL Lysotracker 0,1

Chase 4 h with Leupeptin (10 μg/ml)
0
RFFL facilitates lysosomal delivery of the misfolded CFTR
 AlphaLISA measures the RFFL-ΔF508 NBD1 direct interaction
RFFL-ΔF508 NBD1 interaction Competition assay
(50 nM RFFL & 75 nM NBD1) (50 nM RFFL & 75 nM ΔF508-NBD1)
384 well
120 384 well RFFL ΔRING
140
DR1-2 RING
ΔF508-NBD1 interaction (%)

100 120

ΔF508-NBD1 interaction (%)

80 100

60 80

60
40
40
20
20
0
unfolded folded 0
(42°C, 5 min) 0 0.5 1.5 5 0.5 1.5 5 (μM)

ΔF508 NBD1 BSA RFFL ΔRING

RFFL-NBD1 PPI is RFFL NT fragment inhibits the PPI.

unfolding-dependent as in cells.
Useful for screening of the PPI inhibitor
 Orkambi is good enough for CF therapy ?
Correlation between CFTR activity & CF biomarker
Adopted from Accurso FJ et al, J Cyst Fibros. 2014

100 Non-CF
Hetero
Healthy CF PS
CFTR activity (% of WT)

CF PI
80 DF homo

Further improvement needs for

60 the highly efficacious therapy !!

40
35% function is restored. Modest clinical benefit
Van Goor F et al, PNAS. 2011 (FEV1 < 4% improvement)
20
Wainwright CE et al, N Engl J Med. 2015

0 ΔF508 CF
0 50 100 150
Sweat Cl- (mM) : CF biomarker

Why Orkambi is not good enough ?

 Hepatocytes growth factor (HGF) stimulated Rac1 signaling and contributed to
ΔF508-CFTR anchoring at the cell surface through interactions with NHERF-1.

Selective effect ?

Moniz S et al, ACS Chem Biol. 2013 Feb 15;8(2):432-42.

 GSNOR inhibitor （Cavosonstat: N91115）

http://drug-dev.com/therapeutic-focus-gsnor-inhibition-
Marozkina NV et al, to-stabilize-improve-mutant-cftr-processing/
Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11393-8
 Ubiquitination determines the CFTR elimination from the PM
CFTR ubiquitination CFTR PM stability

ΔF508 mutation facilitates the CFTR Ubiquitin fusion facilitates the CFTR
ubiquitiantion in the post-Golgi. elimination from the PM.

Okiyoneda T et al, Dev Cell 2018

 CHIP is an E3 ligase responsible for the peripheral CFTR QC
CFTR PM stability Mature CFTR stability

HeLa cells CFBE cells

CHIP KD stabilizes rΔF508 CFTR at CHIP KD stabilizes rΔF508 CFTR at

the PM in HeLa cells. the post-Golgi in CFBE cells.

Okiyoneda T et al, Science 2010 Fu L et al, PLoS One 2015

 CFTR mutations and CF drug

T70

Ataluren Orkambi Kalydeco Kalydeco Cavosonstat

Drug
(Lumacaftor (Ivacaftor) (Ivacaftor) CAL inhibitor ?
& Ivacaftor) (CT007)
potentiator
Corrector & potentiator stabilizer
Lopes-Pacheco M. Front Pharmacol. 2016 Sep 5;7:275 (modified)
 Ubiquitination
E1 (2)
Ub activating enzyme

E2 (~40)
Ub conjugating enzyme

E3 (600-1000)
Ub ligase

E3 determines substrate specificity CF drug target

ΔF508 CFTR-specific E3
Jim Kling Nature Biotechnology 28, 1236–1238 (2010)